Pharmaceutical compositions of beta-lapachone and beta-lapachone analogs with improved tumor targeting potential

ABSTRACT

The present invention relates to polymer-modified quinone-containing and carbonyl-containing therapeutic agents, including polymer-modified β-lapachone compounds, and methods of treating cancer by administering the polymer-modified therapeutic agents to a subject. Polymer-modification of therapeutic agents, such as β-lapachone compounds, provides effective transport of polymer-modified therapeutic agents to tumor cells or tumor tissues by exploiting the EPR effect in tumor tissues.

RELATED APPLICATION

The present application is a divisional application of U.S. Ser. No.11/201,097 filed Aug. 11, 2005, which claims the benefit of, andpriority to, U.S. Ser. No. 60/600,373, filed Aug. 11, 2004. The contentsof each of these applications is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to improved compositions and methods fordelivering therapeutic agents bearing carbonyl or quinonefunctionalities and improved methods for treating cancer. One embodimentof the invention relates to improved β-lapachone compositions andmethods of using such compositions for the treatment of cancer.

BACKGROUND OF THE INVENTION

The delivery of therapeutic agents to specific tissues or sites within abody presents a variety of challenges, particularly where local deliveryof a high dose of an insoluble therapeutic agent to a specific tissue isdesired. While it has been recognized that modification of therapeuticagents by conjugation to soluble polymers may aid in their localizeddelivery, this technique has required the presence, or introduction, offunctionalities into the therapeutic agent to accommodate a linkage tothe polymer vehicle. Where it is necessary to introduce functionalityinto a therapeutic agent by chemical modification to accommodate alinkage, characteristics of the therapeutic agent such as potency,half-life, and metabolism may be altered. There is continuing need forpolymer conjugates that can solublize and deliver therapeutic agents tospecific tissues without requiring functionalization of the therapeuticagent to accommodate linkage to the polymer vehicle. Moreover, targetingof therapeutics to specific tissues via linkage to a polymeric deliveryvehicle may result in diminished side effects.

There are insufficient means for conjugation of therapeutic agents viaquinone and carbonyl functionalities to polymeric vehicles that canrelease the therapeutic agent unaltered under in vivo conditions. It maybe advantageous to solubilize and deliver such carbonyl-containing andquinone-containing therapeutic agents to specific tissues by conjugationwith suitable polymer vehicles. Therapeutic agents containing carbonylor quinone functionalities that might advantageously be delivered bysuitable polymeric vehicles include lobeline, acebutolol, methyprylon,haloperidol, molindone, naloxone, oxycodone, methadone, ketanserin,tolmetin, ketoprofen, nabumetone, canrenone, canrenonate, mebendazole,oxolinic acid, tetracycline, chlortetracycline, oxytetracycline,demeclocycline, doxycycline, minocycline, daunorubicin, doxorubicin,mitoxantrone, plicamycin, mitomycin, indan-1,3-dione, anisindione,testosterone (and related C-17 esters, e.g., propionate, enanthate,cypionate), dihydrotesterone, cyproterone acetate, estrone,progesterone, medroxyprogesterone acetate, hydroxyprogesterone caproate,norethindrone, norethynodrel, megestrol acetate, norgestrel,mifepristone, methandrostenolone, oxandrolone, testolactone, cyproteroneacetate, prednisone, prednisolone, betamethasone, dexamethasone, other3-, 17-, or 20-ketosteroids (e.g., dehydroepiandorsterone,androstenedione, cortisol, cortisone, aldosterone, etc.), andparticularly β-lapachone compounds.

β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione),a quinone, is derived from lapachol (a naphthoquinone) which can beisolated from the lapacho tree (Tabebuia avellanedae), a member of thecatalpa family (Bignoniaceae). Lapachol and β-lapachone (with numbering)have the following chemical structures:

β-lapachone, as well as its intermediates, derivatives and analogsthereof, are described in Li, C. J. et al., (1993) J. Biol. Chem.,268(30): 22463-22468.

As a single agent, β-lapachone has demonstrated significantantineoplastic activity against human cancer cell lines atconcentrations typically in the range of 1-10 μM (IC₅₀). Cytotoxicityhas been demonstrated in transformed cell lines derived from: patientswith promyelocytic leukemia (Planchon et al., (1996) Cancer Res., 55:3706-3711), prostate (Li, C. J., et al., (1995) Cancer Res., 55:3712-3715), malignant glioma (Weller, M. et al., (1997) Int. J. Cancer,73: 707-714), hepatoma (Lai, C. C., et al., (1998) Histol Histopathol,13: 89-97), colon (Huang, L., et al., (1999) Mol Med, 5: 711-720),breast (Wuertzberger, S. M., et al., (1998) Cancer Res., 58: 1876),ovarian (Li, C. J. et al., (1999) Proc. Natl. Acad. Sci. USA, 96(23):13369-13374), pancreatic (Li, Y., et al., (2000) Mol Med, 6: 1008-1015;Li, Y., (1999) Mol Med, 5: 232-239), and multiple myeloma cell lines,including drug-resistant lines (Li, Y., (2000) Mol Med, 6: 1008-1015).No cytotoxic effects were observed on normal fresh or proliferatinghuman peripheral blood mononuclear cells (PBMC) (Li, Y., (2000) Mol Med,6: 1008-1015).

β-lapachone appears to work by inducing unscheduled expression ofcheckpoint molecules, e.g., E2F, independent of DNA damage and cellcycle stages. Several studies have shown that β-lapachone activatescheckpoints and induces cell death in cancer cells from a variety oftissues without affecting normal cells from these tissues (U.S. PatentApplication Publication No. 2002/0169135). In normal cells with theirintact regulatory mechanisms, such an imposed expression of a checkpointmolecule results in a transient expression pattern and causes littleconsequence. In contrast, cancer and pre-cancer cells have defectivemechanisms, which result in unchecked and persistent expression ofunscheduled checkpoint molecules, e.g., E2F, leading to selective celldeath in cancer and pre-cancer cells.

β-lapachone has been shown to be a DNA repair inhibitor that sensitizescells to DNA-damaging agents including radiation (Boothman, D. A. etal., Cancer Res, 47 (1987) 5361; Boorstein, R. J., et al., Biochem.Biophys. Commun., 117 (1983) 30). β-lapachone has also shown potent invitro inhibition of human DNA Topoisomerases I (Li, C. J. et al., J.Biol. Chem., 268 (1993) 22463) and II (Frydman, B. et al., Cancer Res.,57 (1997) 620) with novel mechanisms of action. Unlike topoisomerase“poisons” (e.g., camptothecin, etoposide, doxorubicin) which stabilizethe covalent topoisomerase-DNA complex and induce topoisomerase-mediatedDNA cleavage, β-lapachone interacts directly with the enzyme to inhibitcatalysis and block the formation of cleavable complex (Li, C. J. etal., J. Biol. Chem., 268 (1993) 22463), or β-lapachone interacts withthe complex itself, causing religation of DNA breaks and dissociation ofthe enzyme from DNA (Krishnan, P. et al., Biochem Pharm, 60 (2000)1367). β-lapachone and its derivatives have also been synthesized andtested as anti-viral and anti-parasitic agents (Goncalves, A. M., etal., Mol. Biochem. Parasitology, 1 (1980) 167-176; Schaffner-Sabba, K.,et al., J. Med. Chem., 27 (1984) 990-994).

More specifically, β-lapachone appears to work by disrupting DNAreplication, causing cell-cycle delays in G1 and/or S phase, inducingcell death in a wide variety of human carcinoma cell lines without DNAdamage and independent of p53 status (Li, Y. Z. et al. (1999); Huang, L.et al.). Topoisomerase I is an enzyme that unwinds the DNA that makes upthe chromosomes. The chromosomes must be unwound in order for the cellto use the genetic information to synthesize proteins; β-lapachone keepsthe chromosomes wound tight, so that the cell cannot make proteins. As aresult, the cell stops growing. Because cancer cells are constantlyreplicating and circumvent many mechanisms that restrict replication innormal cells, they are more vulnerable to topoisomerase inhibition thanare normal cells.

Another possible intracellular target for β-lapachone in tumor cells isthe enzyme NAP(P)H:quinone oxidoreductase (NQO1). Biochemical studiessuggest that reduction of β-lapachone by NQO1 leads to a “futilecycling” between the quinone and hydroquinone forms with a concomitantloss of reduced NADH or NAD(P)H (Pink, J. J. et al., J. Biol Chem., 275(2000) 5416). The exhaustion of these reduced enzyme cofactors may be acritical factor for the activation of cell death pathways afterβ-lapachone treatment. Reinicke et al. reach a similar conclusion usingmono(arylimino) derivatives of β-lapachone (Reinicke et al., Clin.Cancer. Res. 11(8) (2005) 3055-64).

As a result of these findings, β-lapachone is actively being developedfor the treatment of cancer and tumors. In WO 00/61142, for example,there is disclosed a method and composition for the treatment of cancer,which comprises the administration of an effective amount of a firstcompound, a G1 or S phase drug, such as a β-lapachone, in combinationwith a G2/M drug, such as a taxane derivative. Additionally, U.S. Pat.No. 6,245,807 discloses the use of β-lapachone, amongst otherβ-lapachone derivatives, for use in the treatment of human prostatedisease.

In addition to β-lapachone, a number of β-lapachone analogs havinganti-proliferative properties have been disclosed in the art, such asthose described in PCT International Application PCT/US93/07878 (WO94/04145), and U.S. Pat. No. 6,245,807, in which a variety ofsubstituents may be attached at positions 3- and 4- on the β-lapachonecompound. PCT International Application PCT/US00/10169 (WO 00/61142),discloses β-lapachone, which may have a variety of substituents at the3-position as well as in place of the methyl groups attached at the2-position. U.S. Pat. Nos. 5,763,625, 5,824,700, and 5,969,163 discloseanalogs with a variety of substituents at the 2-, 3- and 4-positions.Furthermore, a number of journals report β-lapachone analogs withsubstituents at one or more of the following positions: 2-, 3-, 8-and/or 9-positions. See, e.g., Sabba et al., (1984) J Med Chem27:990-994 (substituents at the 2-, 8- and 9-positions); Portela andStoppani, (1996) Biochem Pharm 51:275-283 (substituents at the 2- and9-positions); Goncalves et al., (1998) Molecular and BiochemicalParasitology 1:167-176 (substituents at the 2- and 3-positions).

Moreover, structures having sulfur-containing hetero-rings in the “α”and “β” positions of lapachone have been reported (Kurokawa S, (1970)Bulletin of The Chemical Society of Japan 43: 1454-1459; Tapia, R A etal., (2000) Heterocycles 53(3):585-598; Tapia, R A et al., (1997)Tetrahedron Letters 38(1):153-154; Chuang, C P et al., (1996)Heterocycles 40(10):2215-2221; Suginome H et al., (1993) Journal of theChemical Society, Chemical Communications 9:807-809; Tonholo J et al.,(1988) Journal of the Brazilian Chemical Society 9(2):163-169; andKrapcho A P et al., (1990) Journal of Medicinal Chemistry33(9):2651-2655). More particularly, hetero β-lapachone analogs aredisclosed in PCT/US2003/037219, which published as WO 04/045557.

One obstacle to the development of pharmaceutical formulationscomprising β-lapachone or β-lapachone analogs for pharmaceutical use isthe low solubility of β-lapachone compounds in pharmaceuticallyacceptable solvents. β-lapachone compounds are generally highlyinsoluble in water and have only limited solubility in common solventsystems used for pharmaceutical administration, specifically forintravenous delivery of drugs. As a result, there is a need for improvedformulations of β-lapachone compounds for pharmaceutical administration,which are both safe and readily bioavailable to the subject to which theformulation is administered. Importantly, there is an additional need toprovide compositions that target tumor tissue with anti-cancer agentssuch as β-lapachone in such a manner that reduces the potential sideeffects due to the agent being released at unwanted sites. Thisinvention describes systems for the delivery of therapeutic agentshaving carbonyl or quinone functionalities, including β-lapachonecompounds, to tumors in a manner that diminishes unwanted side effects.As the polymeric compositions and methods described herein bindcarbonyl-containing or quinone-containing therapeutic agents through acleavable linkage to the carbonyl or quinone functionalities, they canadvantageously deliver the therapeutic agents without the introductionof additional functionalities that may alter the structure, function,activity, or metabolism of the released therapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides for compositions comprising acarboxyl-containing polymer associated, via a linking agent of formula(I), with one or more carbonyl-containing or quinone-containingtherapeutic agents, wherein said linking agent of formula (I) isassociated with said one or more carbonyl-containing orquinone-containing therapeutic agents by an imine bond;

wherein said linking agent of formula (I) is of the form

wherein

M is selected from the group consisting of: —(C₁-C₈) alkyl-,—(CH₂)_(q)—O—(CH₂)_(r)—, —C(═O)—O—(CH₂)_(r)—, —(C₃-C₇)cycloalkyl-,-aryl-C(═O)—O—(CH₂)_(r)—, —C(═O)—O-aryl-(CH₂)_(r)—,-heteroaryl-C(═O)—O—(CH₂)_(r)—, and —C(═O)—O-heteroaryl-(CH₂)_(r)—;

Z is —OH, a protected amine, or a protected hydroxyl;

each R₁ is independently selected from the group consisting of:hydrogen, halogen, and —(C₁-C₄) alkyl;

q is from 0-6;

r is from 2-6; and

t is from 0-4.

The present invention also provides for pharmaceutical compositionscomprising a therapeutically effective amount of the foregoingcomposition comprising a carboxyl-containing polymer associated, via alinking agent of formula (I), with one or more carbonyl-containing orquinone-containing therapeutic agents, wherein said linking agent offormula (I) is associated with said one or more carbonyl-containing orquinone-containing therapeutic agents by an imine bond. In addition, thepresent invention also contemplates and provides for methods of treatingcancer comprising administering to a subject in need thereof theforegoing pharmaceutical composition comprising a carboxyl-containingpolymer associated, via a linking agent of formula (I), with one or morecarbonyl-containing or quinone-containing therapeutic agents, whereinsaid linking agent of formula (I) is associated with said one or morecarbonyl-containing or quinone-containing therapeutic agents by an iminebond.

The present invention provides for compositions comprising acarboxyl-containing polymer associated, via a linking agent of formula(I), with β-lapachone, wherein said linking agent of formula (I) isassociated with said β-lapachone by an imine bond. The present inventionalso provides for a pharmaceutical composition comprising atherapeutically effective amount of a composition comprising acarboxyl-containing polymer associated, via a linking agent of formula(I), with β-lapachone, wherein said linking agent of formula (I) isassociated with said β-lapachone by an imine bond. In addition, thepresent invention also contemplates and provides for methods of treatingcancer comprising administering to a subject in need thereof any of theforegoing compositions comprising a carboxyl-containing polymerassociated, via a linking agent of formula (I), with β-lapachone,wherein said linking agent of formula (I) is associated with saidβ-lapachone by an imine bond.

The present invention provides for compositions comprising acarboxyl-containing polymer associated, via a linking agent of formula(II), with one or more quinone-containing therapeutic agents, whereinsaid linking agent of formula (II) is associated with said one or morequinone-containing therapeutic agents by a quinol-ester;

wherein said linking agent of formula (II) is of the form

where

each R₃ and R₄ are independently selected from the group consisting ofhydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄) alkyl-aryl, aryl,and heteroaryl;

R₅ is selected from the group consisting of hydrogen, —(C₁-C₈) alkyl,—(C₁-C₈) fluoroalkyl, aryl, and heteroaryl;

R₆ is selected from the group consisting of tert-butoxycarbonyl and CBZ;and

m is from 1 to 8;

alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle.

The present invention also provides for pharmaceutical compositionscomprising a therapeutically effective amount of the foregoingcomposition comprising a carboxyl-containing polymer associated, via alinking agent of formula (II), with one or more quinone-containingtherapeutic agents, wherein said linking agent of formula (II) isassociated with said one or more quinone-containing therapeutic agentsby a quinol-ester. In addition, the present invention also contemplatesand provides for methods of treating cancer comprising administering toa subject in need thereof the foregoing pharmaceutical compositioncomprising a carboxyl-containing polymer associated, via a linking agentof formula (II), with one or more quinone-containing therapeutic agents,wherein said linking agent of formula (II) is associated with said oneor more quinone-containing therapeutic agents by a quinol-ester.

The present invention provides for compositions comprising acarboxyl-containing polymer associated, via a linking agent of formula(II), with β-lapachone, wherein said linking agent of formula (II) isassociated with said β-lapachone by a quinol-ester. The presentinvention also provides for a pharmaceutical composition comprising atherapeutically effective amount of the foregoing composition comprisinga carboxyl-containing polymer associated, via a linking agent of formula(II), with β-lapachone, wherein said linking agent of formula (II) isassociated with said β-lapachone by a quinol-ester. In addition, thepresent invention also contemplates and provides for methods of treatingcancer comprising administering to a subject in need thereof theforegoing pharmaceutical composition comprising a carboxyl-containingpolymer associated, via a linking agent of formula (II), withβ-lapachone, wherein said linking agent of formula (II) is associatedwith said β-lapachone by a quinol-ester.

The present invention provides for compositions comprising acarboxyl-containing polymer associated, via a linking agent of formula(III), with one or more quinone-containing therapeutic agents, whereinsaid linking agent of formula (III) is associated with said one or morequinone-containing therapeutic agents by a ketal linkage;

wherein said linking agent of formula (III) is of the form

where

X is a hydroxyl, a protected hydroxyl or a protected amine;

each of R₇ and R₈ is independently selected from the group consisting ofhydrogen, and (C₁-C₄) alkyl;

R₉ is H; and

p is 1-4.

The present invention also provides for pharmaceutical compositionscomprising a therapeutically effective amount of the foregoingcomposition comprising a carboxyl-containing polymer associated, via alinking agent of formula (III), with one or more quinone-containingtherapeutic agents, wherein said linking agent of formula (III) isassociated with said one or more quinone-containing therapeutic agentsby a ketal linkage. In addition, the present invention also contemplatesand provides for methods of treating cancer comprising administering toa subject in need thereof the foregoing pharmaceutical compositioncomprising a carboxyl-containing polymer associated, via a linking agentof formula (III), with one or more quinone-containing therapeuticagents, wherein said linking agent of formula (III) is associated withsaid one or more quinone-containing therapeutic agents by a ketallinkage.

The present invention provides for compositions comprising acarboxyl-containing polymer associated, via a linking agent of formula(III), with β-lapachone, wherein said linking agent of formula (III) isassociated with said β-lapachone by a ketal linkage. The presentinvention also provides for a pharmaceutical composition comprising atherapeutically effective amount of the foregoing composition comprisinga carboxyl-containing polymer associated, via a linking agent of formula(III), with one or more quinone-containing therapeutic agents, whereinsaid linking agent of formula (III) is associated with said one or morequinone-containing therapeutic agents by a ketal linkage. In addition,the present invention also contemplates and provides for methods oftreating cancer comprising administering to a subject in need thereofthe foregoing pharmaceutical composition comprising acarboxyl-containing polymer associated, via a linking agent of formula(III), with one or more quinone-containing therapeutic agents, whereinsaid linking agent of formula (III) is associated with said one or morequinone-containing therapeutic agents by a ketal linkage.

The present invention provides for methods of preparing apolymer-modified therapeutic agent comprising the steps of:

-   -   (i) providing a linking agent of formula (I):

wherein

M is a spacer group selected from the group consisting of: —(C₁-C₈)alkyl-, —(CH₁₂)_(q)—O—(CH₂)_(r)—, —C(═O)—O—(CH₂)_(r)—,—(C₃-C₇)cycloalkyl-, -aryl-C(═O)—O—(CH₂)_(r)—, —C(═O)—O-aryl-(CH₂)_(r)—,-heteroaryl-C(═O)—O—(CH₂)_(r)—, and —C(═O)—O-heteroaryl-(CH₂)_(r)—;

Z is —OH, a protected amine, or a protected hydroxyl;

each R₁ is independently selected from the group consisting of:hydrogen, halogen, and —(C₁-C₄) alkyl;

q is from 0-6;

r is from 2-6; and

t is from 0-4;

-   -   (ii) providing a quinone-containing therapeutic agent or a        carbonyl-containing therapeutic agent;    -   (iii) contacting said linking agent of formula (I) with said        quinone-containing therapeutic agent or said carbonyl-containing        therapeutic agent under conditions for the formation of an imine        bond there between, resulting in the formation of a linker        conjugated therapeutic agent bearing a Z group;    -   (iv) providing a carboxyl-containing polymer; and    -   (v) contacting said carboxyl-containing polymer with said linker        conjugated therapeutic agent under conditions wherein when said        Z group is a hydroxyl Z forms an ester bond with said        carboxyl-containing polymer, or wherein when said Z group is an        amine Z forms an amide bond with a carboxyl group of said        carboxyl-containing polymer.

In addition, the present invention provides for the products of theforegoing method. Moreover, in an embodiment, the present inventioncontemplates and provides for the products of the foregoing methodwherein the quinone-containing therapeutic agent is mitomycin C,doxorubicin, actinomycin D (dactinomycin), or β-lapachone.

The present invention provides for methods of preparing apolymer-modified therapeutic agent comprising the steps of:

-   -   (i) providing a linking agent of formula (II)

where

each R₃ and R₄ are independently selected from the group consisting ofhydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄) alkyl-aryl, aryl,and heteroaryl;

R₅ is selected from the group consisting of hydrogen, —(C₁-C₈) alkyl,—(C₁-C₈) fluoroalkyl, aryl, and heteroaryl;

R₆ is selected from the group consisting of -tert-butoxycarbonyl andCBZ; and

m is from 1 to 8;

alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle;

-   -   (ii) providing a quinone-containing therapeutic agent;    -   (iii) providing an alkyl carboxylic acid of the formula R₂COOH,        wherein R₂ is (C₁-C₈) alkyl, cycloalkyl, aryl, heterocycle,        heteroaryl, aryl-alkyl, or alkylaryl, and wherein said alkyl and        cycloalkyl groups within R₂ may be substituted with one or more        substituents independently selected from the group consisting of        —H and —F, and wherein said aryl groups within R₂ may be        substituted with one or more substituents independently selected        from the group consisting of —H and halogen;    -   (iv) contacting said linking agent of formula (II) with said        quinone-containing therapeutic agent and said alkyl carboxylic        acid of formula R₂COOH under conditions for the formation of a        quinol-ester linker conjugated therapeutic agent;    -   (v) converting R₆ to hydrogen by deprotection to form a        deprotected quinol-ester linker conjugated therapeutic agent        bearing an amine group;    -   (vi) providing a carboxyl-containing polymer; and    -   (vii) contacting said carboxyl-containing polymer with said        deprotected quinol-ester linker conjugated therapeutic agent        bearing an amine group under conditions where said amine group        forms an amide bond with a carboxyl group of said        carboxyl-containing polymer.

In addition, the present invention provides for the products of theforegoing method. Moreover, in an embodiment, the present inventioncontemplates and provides for the products of the foregoing methodwherein the quinone-containing therapeutic agent is mitomycin C,doxorubicin, actinomycin D (dactinomycin), or β-lapachone.

The present invention provides for methods of preparing apolymer-modified therapeutic agent comprising the steps of:

-   -   (i) providing a linking agent of formula (III)

where

X is a hydroxyl, a protected hydroxyl or a protected amine;

each of R₇ and R₈ is independently selected from the group consisting ofhydrogen, and (C₁-C₄) alkyl;

R₉ is H; and

p is 1-4;

-   -   (ii) providing a quinone-containing or carbonyl-containing        therapeutic agent;    -   (iii) contacting said linking agent of formula (III) with said        quinone-containing or said carbonyl-containing therapeutic agent        under conditions for the formation of a ketal linker conjugated        therapeutic agent;    -   (iv) where X is a protected hydroxyl or a protected amine,        converting it to an amine of the form —NH₂ by deprotection to        form a deprotected ketal linker conjugated therapeutic agent;    -   (v) providing a carboxyl-containing polymer; and    -   (vi) contacting said carboxyl-containing polymer with said ketal        linker conjugated therapeutic agent or said deprotected ketal        linker conjugated therapeutic agent under conditions wherein        when X is a hydroxyl, X forms an ester bond with said        carboxyl-containing polymer, or wherein when X is —NH₂ X forms        an amide bond with said carboxyl-containing polymer.

In addition, the present invention provides for the products of theforegoing method.

Moreover, in an embodiment, the present invention contemplates andprovides for the products of the foregoing method wherein thequinone-containing therapeutic agent is mitomycin C, doxorubicin,actinomycin D (dactinomycin), or β-lapachone.

CERTAIN EMBODIMENTS

The present invention provides for certain embodiments ofcarboxyl-containing polymers wherein said carboxyl-containing polymer isa PGA polymer, and said carboxyl-containing polymer comprises one ormore residues of the form

where “*” indicates the points of attachment to other residues of saidPGA polymer.

The present invention provides for certain embodiments ofcarboxyl-containing polymers wherein said carboxyl-containing polymer isa PGA polymer, and said carboxyl-containing polymer comprises one ormore residues of the form

where “*” indicates the points of attachment to other residues of saidPGA polymer.

The present invention provides for certain embodiments ofcarboxyl-containing polymers wherein said carboxyl-containing polymer isa PGA polymer and said carboxyl-containing polymer comprises one or moreresidues of the form

where “*” indicates the points of attachment to other residues of saidPGA polymer.

The present invention also provides for the following embodiments:

Embodiment 1. A composition comprising a carboxyl-containing polymerassociated, via a linking agent of formula (I), with one or morecarbonyl-containing or quinone-containing therapeutic agents, whereinsaid linking agent of formula (I) is associated with said one or morecarbonyl-containing or quinone-containing therapeutic agents by an iminebond; wherein said linking agent of formula (I) is of the form

wherein

M is selected from the group consisting of: —(C₁-C₈) alkyl-,—(CH₂)_(q)—O—(CH₂)_(r)—, —C(═O)—O—(CH₂)_(r)—, —(C₃-C₇)cycloalkyl-,-aryl-C(═O)—O—(CH₂)_(r)—, —C(═O)—O-aryl-(CH₂)_(r)—,-heteroaryl-C(═O)—O—(CH₂)_(r)—, and —C(═O)—O-heteroaryl-(CH₂)_(r)—;

Z is —OH, a protected amine, or a protected hydroxyl;

each R₁ is independently selected from the group consisting of:hydrogen, halogen, and —(C₁-C₄) alkyl;

q is from 0-6;

r is from 2-6; and

t is from 0-4.

Embodiment 2. The composition of embodiment 1, wherein said one or morecarbonyl-containing or quinone-containing therapeutic agents isβ-lapachone.Embodiment 3. The composition of embodiment 1, wherein saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 4. The composition of embodiment 1, wherein said one or morecarbonyl-containing or quinone-containing therapeutic agents isβ-lapachone, and said carboxyl-containing polymer is a PEG-dicarboxylicacid, SMA, PGA or PAA polymer.Embodiment 5. The composition of embodiment 4, wherein saidcarboxyl-containing polymer is a PGA polymer, and saidcarboxyl-containing polymer comprises one or more residues of the form

where “*” indicates the points of attachment to other residues of saidPGA polymer.Embodiment 6. A pharmaceutical composition comprising a therapeuticallyeffective amount of a composition of any of embodiments 1-5.Embodiment 7. The pharmaceutical composition of embodiment 6, furthercomprising one or more pharmaceutically acceptable excipients.Embodiment 8. The pharmaceutical composition of embodiment 6, whereinsaid pharmaceutical composition is a lyophilized powder.Embodiment 9. The pharmaceutical composition of embodiment 6, whereinsaid pharmaceutical composition is a formulation for oraladministration.Embodiment 10. The pharmaceutical composition of embodiment 6, whereinsaid pharmaceutical composition is a formulation for parenteraladministration.Embodiment 11. The pharmaceutical composition of embodiment 6, whereinsaid pharmaceutical composition is a formulation for intravenousadministration.Embodiment 12. A method of treating cancer comprising administering to asubject in need thereof the pharmaceutical composition of embodiment 6.Embodiment 13. The method of treating cancer of embodiment 12, whereinsaid cancer is lung cancer, breast cancer, colon cancer, ovarian cancer,prostate cancer, multiple myeloma or malignant melanoma.Embodiment 14. The method of treating cancer of embodiment 12, whereinsaid pharmaceutical composition is administered orally.Embodiment 15. The method of treating cancer of embodiment 12, whereinsaid pharmaceutical composition is administered parenterally.Embodiment 16. The method of treating cancer of embodiment 12, whereinsaid pharmaceutical composition is administered intravenously.Embodiment 17. The method of treating cancer of embodiment 12, furthercomprising administering a second anti-cancer agent.Embodiment 18. A composition comprising a carboxyl-containing polymerassociated, via a linking agent of formula (I), with β-lapachone,wherein said linking agent of formula (I) is associated with saidβ-lapachone by an imine bond.Embodiment 19. The composition of embodiment 18, wherein saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 20. A pharmaceutical composition comprising a therapeuticallyeffective amount of a composition of either embodiment 18 or embodiment19.Embodiment 21. The pharmaceutical composition of embodiment 20, furthercomprising one or more pharmaceutically acceptable excipients.Embodiment 22. The pharmaceutical composition of embodiment 20, whereinsaid pharmaceutical composition is a lyophilized powder.Embodiment 23. The pharmaceutical composition of embodiment 20, whereinsaid pharmaceutical composition is a formulation for oraladministration.Embodiment 24. The pharmaceutical composition of embodiment 20, whereinsaid pharmaceutical composition is a formulation for parenteraladministration.Embodiment 25. The pharmaceutical composition of embodiment 20, whereinsaid pharmaceutical composition is a formulation for intravenousadministration.Embodiment 26. A method of treating cancer comprising administering to asubject in need thereof the pharmaceutical composition of embodiment 20.Embodiment 27. The method of treating cancer of embodiment 26, whereinsaid cancer is lung cancer, breast cancer, colon cancer, ovarian cancer,prostate cancer, multiple myeloma or malignant melanoma.Embodiment 28. The method of treating cancer of embodiment 26, whereinsaid pharmaceutical composition is administered orally.Embodiment 29. The method of treating cancer of embodiment 26, whereinsaid pharmaceutical composition is administered parenterally.Embodiment 30. The method of treating cancer of embodiment 26, whereinsaid pharmaceutical composition is administered intravenously.Embodiment 31. The method of treating cancer of embodiment 26, furthercomprising administering a second anti-cancer agent.Embodiment 32. A composition comprising a carboxyl-containing polymerassociated, via a linking agent of formula (II), with one or morequinone-containing therapeutic agents, wherein said linking agent offormula (II) is associated with said one or more quinone-containingtherapeutic agents by a quinol-ester;wherein said linking agent of formula (II) is of the form

where

each R₃ and R₄ are independently selected from the group consisting ofhydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄) alkyl-aryl, aryl,and heteroaryl;

R₅ is selected from the group consisting of hydrogen, —(C₁-C₈) alkyl,—(C₁-C₈) fluoroalkyl, aryl, and heteroaryl;

R₆ is selected from the group consisting of -tert-butoxycarbonyl andCBZ; and

m is from 1 to 8;

alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle.

Embodiment 33. The composition of embodiment 32, wherein said one ormore quinone-containing therapeutic agents is β-lapachone.Embodiment 34. The composition of embodiment 32, wherein saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 35. The composition of embodiment 32, wherein said one ormore quinone-containing therapeutic agents is β-lapachone, and saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 36. The composition of embodiment 32, wherein saidcarboxyl-containing polymer is a PGA polymer, and saidcarboxyl-containing polymer comprises one or more residues of the form

where “*” indicates the points of attachment to other residues of saidPGA polymer.Embodiment 37. A pharmaceutical composition comprising a therapeuticallyeffective amount of a composition of any of embodiments 32-36.Embodiment 38. The pharmaceutical composition of embodiment 37, furthercomprising one or more pharmaceutically acceptable excipients.Embodiment 39. The pharmaceutical composition of embodiment 37, whereinsaid pharmaceutical composition is a lyophilized powder.Embodiment 40. The pharmaceutical composition of embodiment 37, whereinsaid pharmaceutical composition is a formulation for oraladministration.Embodiment 41. The pharmaceutical composition of embodiment 37, whereinsaid pharmaceutical composition is a formulation for parenteraladministration.Embodiment 42. The pharmaceutical composition of embodiment 37, whereinsaid pharmaceutical composition is a formulation for intravenousadministration.Embodiment 43. A method of treating cancer comprising administering to asubject in need thereof the pharmaceutical composition of embodiment 37.Embodiment 44. The method of treating cancer of embodiment 43, whereinsaid cancer is lung cancer, breast cancer, colon cancer, ovarian cancer,prostate cancer, multiple myeloma or malignant melanoma.Embodiment 45. The method of treating cancer of embodiment 43, whereinsaid pharmaceutical composition is administered orally.Embodiment 46. The method of treating cancer of embodiment 43, whereinsaid pharmaceutical composition is administered parenterally.Embodiment 47. The method of treating cancer of embodiment 43, whereinsaid pharmaceutical composition is administered intravenously.Embodiment 48. The method of treating cancer of embodiment 43, furthercomprising administering a second anti-cancer agent.Embodiment 49. A composition comprising a carboxyl-containing polymerassociated, via a linking agent of formula (II), with β-lapachone,wherein said linking agent of formula (II) is associated with saidβ-lapachone by a quinol-ester.Embodiment 50. The composition of embodiment 49, wherein saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 51. A pharmaceutical composition comprising a therapeuticallyeffective amount of a composition of either embodiment 49 or embodiment50.Embodiment 52. The pharmaceutical composition of embodiment 51, furthercomprising one or more pharmaceutically acceptable excipients.Embodiment 53. The pharmaceutical composition of embodiment 51, whereinsaid pharmaceutical composition is a lyophilized powder.Embodiment 54. The pharmaceutical composition of embodiment 51, whereinsaid pharmaceutical composition is a formulation for oraladministration.Embodiment 55. The pharmaceutical composition of embodiment 51, whereinsaid pharmaceutical composition is a formulation for parenteraladministration.Embodiment 56. The pharmaceutical composition of embodiment 51, whereinsaid pharmaceutical composition is a formulation for intravenousadministration.Embodiment 57. A method of treating cancer comprising administering to asubject in need thereof the pharmaceutical composition of embodiment 51.Embodiment 58. The method of treating cancer of embodiment 57, whereinsaid cancer is lung cancer, breast cancer, colon cancer, ovarian cancer,prostate cancer, multiple myeloma or malignant melanoma.Embodiment 59. The method of treating cancer of embodiment 57, whereinsaid pharmaceutical composition is administered orally.Embodiment 60. The method of treating cancer of embodiment 57, whereinsaid pharmaceutical composition is administered parenterally.Embodiment 61. The method of treating cancer of embodiment 57, whereinsaid pharmaceutical composition is administered intravenously.Embodiment 62. The method of treating cancer of embodiment 57, furthercomprising administering a second anti-cancer agent.Embodiment 63. A composition comprising a carboxyl-containing polymerassociated, via a linking agent of formula (III), with one or morequinone-containing therapeutic agents, wherein said linking agent offormula (III) is associated with said one or more quinone-containingtherapeutic agents by a ketal linkage;wherein said linking agent of formula (III) is of the form

where

X is a hydroxyl, a protected hydroxyl or a protected amine;

each of R₇ and R₈ is independently selected from the group consisting ofhydrogen, and (C₁-C₄) alkyl;

R₉ is H; and

p is 1-4.

Embodiment 64. The composition of embodiment 63, wherein said one ormore quinone-containing therapeutic agents is β-lapachone.Embodiment 65. The composition of embodiment 63, wherein saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 66. The composition of embodiment 63, wherein said one ormore quinone-containing therapeutic agents is β-lapachone, and saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 67. The composition of embodiment 66, wherein saidcarboxyl-containing polymer is a PGA polymer, and saidcarboxyl-containing polymer comprises one or more residues of the form

where “*” indicates the points of attachment to other residues of saidPGA polymer.Embodiment 68. A pharmaceutical composition comprising a therapeuticallyeffective amount of a composition of any of embodiments 63-67.Embodiment 69. The pharmaceutical composition of embodiment 68, furthercomprising one or more pharmaceutically acceptable excipients.Embodiment 70. The pharmaceutical composition of embodiment 68, whereinsaid pharmaceutical composition is a lyophilized powder.Embodiment 71. The pharmaceutical composition of embodiment 68, whereinsaid pharmaceutical composition is a formulation for oraladministration.Embodiment 72. The pharmaceutical composition of embodiment 68, whereinsaid pharmaceutical composition is a formulation for parenteraladministration.Embodiment 73. The pharmaceutical composition of embodiment 68, whereinsaid pharmaceutical composition is a formulation for intravenousadministration.Embodiment 74. A method of treating cancer comprising administering to asubject in need thereof the pharmaceutical composition of embodiment 68.Embodiment 75. The method of treating cancer of embodiment 74, whereinsaid cancer is lung cancer, breast cancer, colon cancer, ovarian cancer,prostate cancer, multiple myeloma or malignant melanoma.Embodiment 76. The method of treating cancer of embodiment 74, whereinsaid pharmaceutical composition is administered orally.Embodiment 77. The method of treating cancer of embodiment 74, whereinsaid pharmaceutical composition is administered parenterally.Embodiment 78. The method of treating cancer of embodiment 74, whereinsaid pharmaceutical composition is administered intravenously.Embodiment 79. The method of treating cancer of embodiment 74, furthercomprising administering a second anti-cancer agent.Embodiment 80. A composition comprising a carboxyl-containing polymerassociated, via a linking agent of formula (III), with β-lapachone,wherein said linking agent of formula (III) is associated with saidβ-lapachone by a ketal linkage.Embodiment 81. The composition of embodiment 80, wherein saidcarboxyl-containing polymer is a PEG-dicarboxylic acid, SMA, PGA or PAApolymer.Embodiment 82. A pharmaceutical composition comprising a therapeuticallyeffective amount of a composition of either embodiment 80 or embodiment81.Embodiment 83. The pharmaceutical composition of embodiment 82, furthercomprising one or more pharmaceutically acceptable excipients.Embodiment 84. The pharmaceutical composition of embodiment 82, whereinsaid pharmaceutical composition is a lyophilized powder.Embodiment 85. The pharmaceutical composition of embodiment 82, whereinsaid pharmaceutical composition is a formulation for oraladministration.Embodiment 86. The pharmaceutical composition of embodiment 82, whereinsaid pharmaceutical composition is a formulation for parenteraladministration.Embodiment 87. The pharmaceutical composition of embodiment 82, whereinsaid pharmaceutical composition is a formulation for intravenousadministration.Embodiment 88. A method of treating cancer comprising administering to asubject in need thereof the pharmaceutical composition of embodiment 82.Embodiment 89. The method of treating cancer of embodiment 88, whereinsaid cancer is lung cancer, breast cancer, colon cancer, ovarian cancer,prostate cancer, multiple myeloma or malignant melanoma.Embodiment 90. The method of treating cancer of embodiment 88, whereinsaid pharmaceutical composition is administered orally.Embodiment 91. The method of treating cancer of embodiment 88, whereinsaid pharmaceutical composition is administered parenterally.Embodiment 92. The method of treating cancer of embodiment 88, whereinsaid pharmaceutical composition is administered intravenously.Embodiment 93. The method of treating cancer of embodiment 88, furthercomprising administering a second anti-cancer agent.Embodiment 94. A method of preparing a polymer-modified therapeuticagent comprising the steps of:

-   -   (i) providing a linking agent of formula (I):

wherein

M is a spacer group selected from the group consisting of: —(C₁-C₈)alkyl-, —(C₂)_(q)—O—(CH₂)_(r)—, —C(═O)—O—(CH₂)_(r)—,—(C₃-C₇)cycloalkyl-, -aryl-C(═O)—O—(CH₂)_(r)—, —C(═O)—O-aryl-(CH₂)_(r)—,-heteroaryl-C(═O)—O—(CH₂)_(r)—, and —C(═O)—O-heteroaryl-(CH₂)_(r)—;

Z is —OH, a protected amine, or a protected hydroxyl;

each R₁ is independently selected from the group consisting of:hydrogen, halogen, and —(C₁-C₄) alkyl;

q is from 0-6;

r is from 2-6; and

t is from 0-4;

-   -   (ii) providing a quinone-containing therapeutic agent or a        carbonyl-containing therapeutic agent;    -   (iii) contacting said linking agent of formula (I) with said        quinone-containing therapeutic agent or said carbonyl-containing        therapeutic agent under conditions for the formation of an imine        bond there between, resulting in the formation of a linker        conjugated therapeutic agent bearing a Z group;    -   (iv) providing a carboxyl-containing polymer; and    -   (v) contacting said carboxyl-containing polymer with said linker        conjugated therapeutic agent under conditions wherein when said        Z group is a hydroxyl Z forms an ester bond with said        carboxyl-containing polymer, or wherein when said Z group is an        amine Z forms an amide bond with a carboxyl group of said        carboxyl-containing polymer.        Embodiment 95. The method of embodiment 94, wherein when said Z        group of said linker conjugated therapeutic agent is a protected        amine, or a protected hydroxyl, said protected amine, or        protected hydroxyl is deprotected prior to contacting said        carboxyl-containing polymer with said linker conjugated        therapeutic agent in step (v).        Embodiment 96. The method of embodiments 94 or 95, where said        carboxyl-containing polymer is a PEG-dicarboxylic acid, PGA, PAA        or SMA polymer.        Embodiment 97. The product produced by the method of embodiments        94 or 95.        Embodiment 98. The product produced by the method of embodiments        94 or 95, wherein said quinone-containing therapeutic agent is        mitomycin C, doxorubicin, actinomycin D (dactinomycin), or        β-lapachone.        Embodiment 99. The product produced by the method of embodiment        94 or 95 wherein said quinone-containing therapeutic agent is        β-lapachone.        Embodiment 100. The product produced by the method of embodiment        97, where said carboxyl-containing polymer is a PEG-dicarboxylic        acid, PGA, PAA or SMA polymer.        Embodiment 101. The product produced by the method of embodiment        98, where said carboxyl-containing polymer is a PEG-dicarboxylic        acid, PGA, PAA or SMA polymer.        Embodiment 102. The product produced by the method of embodiment        99, where said carboxyl-containing polymer is a PEG-dicarboxylic        acid, PGA, PAA or SMA polymer.        Embodiment 103. A method of preparing a polymer-modified        therapeutic agent comprising the steps of:    -   (i) providing a linking agent of formula (II)

where

each R₃ and R₄ are independently selected from the group consisting ofhydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄) alkyl-aryl, aryl,and heteroaryl;

R₅ is selected from the group consisting of hydrogen, —(C₁-C₈) alkyl,—(C₁-C₈) fluoroalkyl, aryl, and heteroaryl;

R₆ is selected from the group consisting of -tert-butoxycarbonyl andCBZ; and

m is from 1 to 8;

alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle;

-   -   (ii) providing a quinone-containing therapeutic agent;    -   (iii) providing an alkyl carboxylic acid of the formula R₂COOH,        wherein R₂ is (C₁-C₈) alkyl, cycloalkyl, aryl, heterocycle,        heteroaryl, aryl-alkyl, or alkylaryl, and wherein said alkyl and        cycloalkyl groups within R₂ may be substituted with one or more        substituents independently selected from the group consisting of        —H and —F, and wherein said aryl groups within R₂ may be        substituted with one or more substituents independently selected        from the group consisting of —H and halogen;    -   (iv) contacting said linking agent of formula (II) with said        quinone-containing therapeutic agent and said alkyl carboxylic        acid of formula R₂COOH under conditions for the formation of a        quinol-ester linker conjugated therapeutic agent;    -   (v) converting R₆ to hydrogen by deprotection to form a        deprotected quinol-ester linker conjugated therapeutic agent        bearing an amine group;    -   (vi) providing a carboxyl-containing polymer; and    -   (vii) contacting said carboxyl-containing polymer with said        deprotected quinol-ester linker conjugated therapeutic agent        bearing an amine group under conditions where said amine group        forms an amide bond with a carboxyl group of said        carboxyl-containing polymer.        Embodiment 104. The method of embodiment 103, where said        carboxyl-containing polymer is a PEG-dicarboxylic acid, PGA, PAA        or SMA polymer.        Embodiment 105. The product produced by the method of        embodiments 103 or 104.        Embodiment 106. The product produced by the method of        embodiments 103 or 104, wherein said quinone-containing        therapeutic agent is mitomycin C, doxorubicin, actinomycin D        (dactinomycin), or β-lapachone.        Embodiment 107. The product produced by the method of        embodiments 103 or 104, wherein said quinone-containing        therapeutic agent is β-lapachone.        Embodiment 108. A method of preparing a polymer-modified        therapeutic agent comprising the steps of:    -   (i) providing a linking agent of formula (III)

where

X is a hydroxyl, a protected hydroxyl or a protected amine;

each of R₇ and R₈ is independently selected from the group consisting ofhydrogen, and (C₁-C₄) alkyl;

R₉ is H; and

p is 1-4;

-   -   (ii) providing a quinone-containing or carbonyl-containing        therapeutic agent;    -   (iii) contacting said linking agent of formula (III) with said        quinone-containing or said carbonyl-containing therapeutic agent        under conditions for the formation of a ketal linker conjugated        therapeutic agent;    -   (iv) where X is a protected hydroxyl or a protected amine,        converting it an amine of the form —NH₂ by deprotection to form        a deprotected ketal linker conjugated therapeutic agent;    -   (v) providing a carboxyl-containing polymer; and    -   (vi) contacting said carboxyl-containing polymer with said ketal        linker conjugated therapeutic agent or said deprotected ketal        linker conjugated therapeutic agent under conditions wherein        when X is a hydroxyl, X forms an ester bond with said        carboxyl-containing polymer, or wherein when X is —NH₂ X forms        an amide bond with said carboxyl-containing polymer.        Embodiment 109. The method of embodiment 108, where said        carboxyl-containing polymer is a PEG-dicarboxylic acid, PGA, PAA        or SMA polymer.        Embodiment 110. The product produced by the method of        embodiments 108 or 109.        Embodiment 111. The product produced by the method of        embodiments 108 or 109, wherein said quinone-containing        therapeutic agent is mitomycin C, doxorubicin, actinomycin D        (dactinomycin), or β-lapachone.        Embodiment 112. The product produced by the method of        embodiments 108 or 109, wherein said quinone-containing        therapeutic agent is β-lapachone.        Embodiment 113. A method for increasing the stability of a        quinone-containing therapeutic agent in serum comprising        administering to a subject in need thereof a therapeutically        effective amount of a composition of embodiments 1, 18, 32, 49,        63 or 80 comprising a polymer-modified quinone-containing        therapeutic agent, wherein the stability of said        quinone-containing therapeutic agent in said serum is increased.        Embodiment 114. A method for increasing the concentration of a        quinone-containing therapeutic agent in serum comprising        administering to a subject in need thereof a therapeutically        effective amount of a composition of embodiments 1, 18, 32, 49,        63 or 80 comprising a polymer-modified quinone-containing        therapeutic agent, wherein the concentration of said        quinone-containing therapeutic agent in said serum is increased.        Embodiment 115. A method for delivering a quinone-containing        therapeutic agent or carbonyl-containing therapeutic agent to a        tumor tissue comprising administering to a subject in need        thereof a therapeutically effective amount of a composition of        embodiments 1, 18, 32, 49, 63 or 80 comprising a        polymer-modified quinone-containing therapeutic agent or a        polymer-modified carbonyl-containing therapeutic agent.        Embodiment 116. The method of embodiment 115, where said        composition comprises a quinone-containing therapeutic agent.        Embodiment 117. The method of embodiment 116, where said        composition comprises a carbonyl-containing therapeutic agent.        Embodiment 118. The method of embodiments 115, 116, or 117,        wherein the half-life of said quinone-containing therapeutic        agent or carbonyl-containing therapeutic agent is increased in        said tumor tissue as compared to the half-life of said        quinone-containing therapeutic agent or carbonyl-containing        therapeutic agent administered as an intravenous bolus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth measurements of the effects of β-lapachone(2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione) (in DMSO) (•),and the polymer-modified β-lapachone compositions5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate (in PEG/water) (♦) and(2,2-dimethyl-5-oxo-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′-yl)methylγ-poly-L-glutamate sodium (in water) (▴) on the proliferation of DLD-1cells and NCM-460 cells at 20 and 72 hours. Panel A indicates theeffects on DLD-1 cells at 20 hours, Panel B indicates the effects onDLD-1 cells at 72 hours, Panel C indicates the effects on NCM-460 cellsat 20 hours, and Panel D indicates the effects on NCM-460 cells at 72hours. Estimates of the IC₅₀ (μM) are given in Table 2. Experimentaldetails are set forth in Example 12.

FIG. 2, Panel A sets forth the release of free β-lapachone(2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione) from5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate (Table 1: Sample B), Panel B sets forth the releaseof free β-lapachone from(2,2-dimethyl-5-oxo-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′-yl)methylγ-poly-L-glutamate sodium (Table 1: Sample J), Panel C sets forth therelease of free β-lapachone fromN-{2-[(4-{[(6Z)-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]-chromen-6(5H)-ylidene]amino}benzoyl)oxy]ethyl}γ-poly-L-glutamate(Table 1: Sample B), and Panel D sets forth the release of freeβ-lapachone from(2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione) from5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate (Table 1: Sample D) in control buffer and in plasma.Cleavage of β-lapachone from the polymer conjugates in control bufferand in plasma is conducted for the indicated times (T) given in hours(h). Control acid (HCl) cleavage of the polyglutamic conjugates inbuffer is indicated by the results labeled “Acid C.” Control acid (HCl)cleavage of the polyglutamic conjugate in plasma is indicated by theresults labeled “Acid pl.” Control base (NaOH) cleavage of thepolyglutamic conjugates in buffer is indicated by the results labeled“base C.” Control base (NaOH) cleavage of the polyglutamic conjugate inplasma is indicated by the results labeled “base pl,” and controlstandard of β-lapachone is labeled “std.” The response factor indicatesthe relative instrument response to β-lapachone, and values associatedwith the “*” indicate the theoretical β-lapachone concentrations basedupon the percentage loading of the respective conjugates. Experimentaldetails are set forth in Examples 13 and 14.

FIG. 3 sets forth a time course of tumor and plasma levels(concentration given in ng/ml) of β-lapachone following administrationof: β-lapachone (reference) by intraperitoneal administration (ip) (60mg/kg) or 5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylglycinate γ-poly-L-glutamate (Table 1: Sample B) by intravenous (iv)administration (26 mg/kg equivalents of β-lapachone, 139 mg/kg of thepolymer conjugate having 19.3% β-lapachone by weight) to Ncr NU/NU tumorbearing mice. Free β-lapachone (♦) in tumors; total β-lapachone (▪) intumors; free β-lapachone in plasma (▴); total β-lapachone in plasma (x);β-lapachone reference tumor (- - ▪ - -); and β-lapachone referenceplasma (• • • • • • •). Experimental details are set forth in Examples13 and 15.

FIG. 4 sets forth a time course of tumor and plasma levels(concentration given in ng/ml) of: β-lapachone following administrationof: β-lapachone (reference) by intraperitoneal administration (ip) (60mg/kg) or(2,2-dimethyl-5-oxo-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′-yl)methylγ-poly-L-glutamate sodium (Table 1: Sample J) by intravenous (iv)administration (60 mg/kg equivalents of β-lapachone, 370 mg/kg of thepolymer conjugate having 16.2% β-lapachone by weight) to Ncr NU/NU tumorbearing mice. Free β-lapachone tumors (♦) free ketal (i.e.4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one)in tumor (▪)(β-lapachone equivalents); free β-lapachone in plasma (Δ);free ketal in plasma (x) (β-lapachone equivalents); β-lapachonereference plasma (- - ▪ - -); and β-lapachone reference tumor (• • • • •• •). Experimental details are set forth in Example 13 and 15.

FIGS. 5A & 5B set forth time courses of tumor (Δ) and plasma (B) levels(concentration given in ng/ml) of β-lapachone following administrationof: β-lapachone (reference) by intraperitoneal administration (ip) (60mg/kg), 5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylglycinate γ-poly-L-glutamate (Table 1: Sample B) by intravenous (iv)administration (14 mg/kg equivalents of β-lapachone, 73 mg/kg of thepolymer conjugate having 19.3% β-lapachone by weight) or5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate (Table 1: Sample D) by intravenous (iv)administration (14 mg/kg equivalents of β-lapachone, 75 mg/kg of thepolymer conjugate having 18.6% β-lapachone by weight) to Ncr NU/NU tumorbearing mice. FIG. 5A: free β-lapachone (▪ and ▴, Table 1: Samples B andD, respectively) in tumors; total β-lapachone (□ and Δ, Table 1: SamplesB and D, respectively) in tumors; β-lapachone reference tumor (♦). FIG.5B: free β-lapachone (▪ and ▴, Table 1: Samples B and D, respectively)in plasma; total β-lapachone (F and A, Table 1: Samples B and D,respectively) in plasma; and β-lapachone reference plasma ♦).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the effective solubilization and transport ofpolymer-modified carbonyl-containing or quinone-containing therapeuticagents and their use in the treatment of diseases, including cancers, bywater-soluble carboxyl-containing polymers. Another aspect of theinvention regards the effective solubilization and transport ofpolymer-modified anti-cancer agents, such as β-lapachone compounds, totumor cells or tumor tissues so as to promote therapeutic effectiveness.

High molecular weight substances, fine particles, fats and oils are moreeasily accumulated and stay for a longer time in tumor sites andinflammatory sites than in normal tissues. As such, long-circulatedmacromolecules, including polypeptide/polymer conjugates, are capable ofspontaneous accumulation in solid tumors. This accumulation may resultfrom an effect termed the enhanced permeability and retention (EPR)effect. Delivery of anti-cancer agents to tumor cells or tumor tissuesin polymer modified forms may advantageously exploit the EPR effect intumor tissues.

Without intending to be limited by theory, the EPR effect is based onthe fact that tumor vasculature, unlike the vasculature of normaltissues, is permeable to macromolecules with a relatively high molecularweight. Structurally, tumor vessels show increased vascular densitycaused by angiogenesis, impaired lymphatic recovery, and lack of asmooth muscle layer in solid tumor vessels. More importantly, functionalaugmentation of this enhanced vascular permeability in tumor tissues hasbeen recognized and includes extensive production of vascular mediatorssuch as bradykinin, nitric oxide, prostaglandins, matrixmetalloprotinases, and vascular endothelial growth factor/vascularpermeability factor, among others. High permeability of the vasculatureallows macromolecules to enter the tumor interstitial space, whilecompromised lymphatic filtration allows them to stay there. Unlikemacromolecules, low-molecular-weight pharmaceuticals are generally notretained in tumors because of their ability to return to the circulationby diffusion. Thus, the conjugation of low molecular weight moleculeswith molecular weight increasing compounds yields complexes that mayspontaneously accumulate in solid tumors.

The linking agents and polymer vehicles employed to prepare the polymerconjugates described herein advantageously permit the selection ofdelivery conditions for carbonyl-containing and quinone-containingtherapeutic agents having a broad range of solubilities and potenciesthrough the choice of linking chemistry (e.g., imine, quinol-ester, orketal), the specific linking agents, and the polymeric vehicle employed.Where the therapeutic agents are of high potency, the present inventionadvantageously permits delivery over short or long time intervals,depending upon the selection of the specific linking agent and polymericvehicle. At the other end of the potency spectrum, the linkingchemistries of the present invention advantageously permit the deliveryof lower potency therapeutic agents over short time intervals, therebypermitting a pharmacologically active amount of the unconjugatedtherapeutic agent to be delivered. In preferred embodiments, a lowpotency therapeutic agent may be maintained at a pharmacologicallyactive level, as measured in either in the plasma or at a site of actionsuch as a tumor, for a time period longer than that which can beachieved by delivery of a bolus of the free therapeutic agent (i.e.,therapeutic agent which is not in a polymer conjugate or prodrug form).In some embodiments, greater than 50% of a therapeutic agent present ina polymer conjugate may be released from the polymer conjugate in 24hours. In other embodiments, greater than 70% of a therapeutic agent maybe released from a polymer conjugate in 24 hours, and in otherembodiments, greater than 90% of a therapeutic agent may be releasedfrom a polymer conjugate in 24 hours. In addition, the relatively highloading of therapeutic agents into the polymer conjugates of theinvention permits delivery of large amounts of the therapeutic agentswhile minimizing the amount, and thus the potential impact, of thepolymeric vehicle required to effectuate the delivery of thetherapeutic.

The ability to select conditions suitable for the delivery oftherapeutic agents, even those having even low solubility in aqueoussolution and low potency, is illustrated by the polymer conjugates ofβ-lapachone described for example in FIGS. 3, 5A and B. Therapeuticlevels of β-lapachone in both tumors and plasma persist for up to about24 hours following the administration to mice of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate (Table 1: Samples B and D). Thus,5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate represents a preferred means of deliveringβ-lapachone because it advantageously increases the solubility,increases the levels achievable and maintainable in both plasma and intumor tissues, and the duration of action relative to the unconjugatedcompound. In addition, the administration of polymer conjugates thatrelease therapeutic over a suitable long time period, such as5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate, permits fewer administrations of the therapeuticagent where the therapeutic is delivered in bolus form. Moreover, thehigh loading of therapeutic that can be achieved using polymerconjugates such as5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate (Table 1: Sample D) advantageously minimizes theamount of excipients that need to be administered to achievepharmacologically effective administration of therapeutic agents,particularly those having low solubility and low potency such asβ-lapachone. For at least these reasons,5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate represents a preferred means of deliveringβ-lapachone as a therapeutic agent.

Another aspect of this invention relates to the unexpected stabilizationobserved for quinone-containing therapeutic agents and carbonylcontaining therapeutic agents presented in the polymer-modified formsdescribed herein. This is reflected in the prolonged half-life of thecompounds both in vitro at neutral pH and in vivo. Related to theincreased stability of quinone-containing therapeutic agents andcarbonyl containing therapeutic agents presented in the polymer-modifiedforms described herein is the higher levels of the drugs that can beboth achieved and maintained in a subject or in specific tissues orplasma. The increased levels of therapeutic agents that may be achievedis particularly important for drugs, such as β-lapachone, whosesolubility might otherwise limit their value as a therapeutics agentsdue to the difficulty of maintaining an effective therapeutic level ofthe drug in the relevant tissues. Maintaining increased levels of suchtherapeutics in tumors is expected to provide a better treatment profilefor cancer patients.

Other aspects of the invention relate to polymer-modified β-lapachonecompounds and methods of treating cancer by administering thepolymer-modified β-lapachone compounds of the invention to a subject, asdescribed in more detail herein. More specifically, the polymer-modifiedβ-lapachone compounds of the invention generally comprise a β-lapachoneor an analog thereof associated with at least one polymeric material byway of one of the linking mechanisms (imine, quinol-ester, or ketal).

Therapeutics may be released from the polymer conjugates of thisinvention substantially by two means, further described below, both ofwhich involve the cleavage of a linking agent bond. The skilled artisanwill recognize that cleavage of the bond between the linking agent andthe polymer or therapeutic agent may result in the production of amodified form of the linking agent. One exemplary modification thatmight occur to a linking agent is one in which the atom of alinker-conjugated therapeutic agent that is initially involved in thebond with the polymeric vehicle is attached to a protective group thatbecomes eliminated in the formation of the polymer conjugate. Uponcleavage from the polymer vehicle, another substituent, such as ahydrogen or hydroxyl group from hydrolysis, would replace the protectivegroup.

The first means of therapeutic agent release from the polymer conjugatesof the invention occurs where the bond between the polymeric vehicle andthe linker is cleaved to release a linker-conjugated therapeutic agent,which like the polymer conjugate, may be considered a prodrug. Where thelinking agent has become modified by the cleavage of the bond betweenthe polymeric vehicle and the linker, the linker-conjugated therapeuticagent released from the polymeric vehicle may be a modified form of thelinker-conjugated therapeutic agent that was used to prepare the polymerconjugate. Subsequent release of the therapeutic agent from the linkingagent or a modified form of the linking agent, may involve not only thecleavage of the therapeutic agent from the linking agent, but also otherenzymatic and non-enzymatic steps. Thus, for example, aquinone-containing therapeutic agent, such as β-lapachone, may bereleased from the polymeric vehicle of a quinol-ester polymer conjugateby cleavage of the bond between the polymeric vehicle and thequinol-ester linker conjugated therapeutic agent. Subsequent release ofa therapeutic agent, such as β-lapachone, from the linking agent may notonly involve the enzymatic or non-enzymatic cleavage of the linkingagent from the quinol-ester, but also other enzymatic or non-enzymaticprocesses. These processes may include, but are not limited to,cleavage, such as a cleavage of a carboxylic acid bound to a hydroxyl ofa quinol-ester (e.g., R₂COOH of quinol-ester conjugates) and theoxidation of the quinol form of a therapeutic, such as β-lapachone, toits quinone form.

The second means of therapeutic agent release from the polymerconjugates of the invention involves the cleavage of the linkage betweenthe linking agent and the therapeutic agent without, or prior to, thecleavage of the bond between the linking agent and the polymer vehicle.As with the first means of therapeutic agent delivery from the polymerconjugates of the invention, release of the therapeutic agent by thesecond means may also involve enzymatic or non-enzymatic processes thatoccur subsequent to the cleavage of the linkage between the linkingagent and the therapeutic agent. Those enzymatic or non-enzymaticprocesses similarly may include, but are not limited to, cleavage of thecarboxylic acid bound to a hydroxyl of a quinol-ester and the oxidationof the quinol form of a therapeutic to the quinone form, such as mayoccur where the therapeutic agent is β-lapachone. Where such enzymaticor non-enzymatic processes occur subsequent to the cleavage of thelinkage between the linking agent and the therapeutic agent, thetherapeutic agent can be considered to be released as a prodrug.

A skilled artisan will also recognize that therapeutic agents present inpolymer conjugates may be subject to enzymatic or non-enzymaticprocesses prior to, or concurrent with, their release from polymerconjugates. Where the enzymatic or non-enzymatic processes modifypolymer conjugates prior to the release of therapeutic agents from thepolymer vehicle, the therapeutic agent may still be released in aprodrug form if the bond between the polymeric vehicle and the linker iscleaved to release a linker-conjugated therapeutic agent. In contrast,whether or not a therapeutic agent is released in a prodrug form wherethe linkage between the linking agent and the therapeutic agent iscleaved without, or prior to, the cleavage of the bond between thelinking agent and the polymer vehicle, depends upon whether the agentreleased upon cleavage of the linkage requires conversion by enzymaticor non-enzymatic means to the active therapeutic. Thus, for example,polymer conjugates containing β-lapachone bound in a quinol-ester mayundergo cleavage of the carboxylic acid associated with one hydroxyl ofthe quinol ester of prior to, concurrent with, or subsequent to cleavageof the linkage between β-lapachone and the linker.

Standard synthetic methods and procedures for the preparation of organicmolecules and functional group transformations and manipulationsincluding the use of protective groups can be obtained from the relevantscientific literature or from standard reference textbooks in the field.Although not limited to any one or several sources, recognized referencetextbooks of organic synthesis include: Smith, M. B.; March, J. March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5^(th)ed.; John Wiley & Sons: New York, 2001; and Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 3^(rd); John Wiley & Sons:New York, 1999. The following descriptions of synthetic methods aredesigned to illustrate, but not limit, general procedures for thepreparation of compounds of the invention.

A. DEFINITIONS

The term “alkyl” refers to radicals containing carbon and hydrogen,without unsaturation. Alkyl radicals can be straight or branched.Exemplary alkyl radicals include, without limitation, methyl, ethyl,propyl, isopropyl, hexyl, t-butyl, sec-butyl and the like. Alkyl groupsmay be denoted by a range, thus, for example, a (C₁-C₆) alkyl group isan alkyl group having from one to six carbon atoms in the straight orbranched alkyl backbone. For example, substituted and unsubstitutedalkyl groups may independently be (C₁-C₅) alkyl, (C₁-C₆) alkyl, (C₁-C₁₀)alkyl, (C₃-C₁₀) alkyl, or (C₅-C₁₀) alkyl. Unless expressly stated, theterm “alkyl” does not include “cycloalkyl.”

A “cycloalkyl” group refers to a cyclic alkyl group having the indicatednumber of carbon atoms in the “ring portion,” where the “ring portion”may consist of one or more ring structures either as fused, spiro, orbridged ring structures. For example, a C₃ to C₆ cycloalkyl group (e.g.,(C₃-C₆) cycloalkyl) is a ring structure having between 3 and 6 carbonatoms in the ring. When no range is given, then cycloalkyl has betweenthree and nine carbon atoms ((C₃-C₉)cycloalkyl) in the ring portion.Exemplary cycloalkyl groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and adamantyl.Preferred cycloalkyl groups have three, four, five, six, seven, eight,nine, or from four to nine carbon atoms in the ring structure.

The term “aryl” refers to an aromatic carbocyclic group, having one,two, or three aromatic rings. Exemplary aryl groups include, withoutlimitation, phenyl, naphthyl, and the like. Aryl groups may be fused toform one or more additional nonaromatic carbocyclic or heterocyclicrings having from 4-9 members. Examples of fused aryl groups includebenzocyclobutanyl, indanyl, tetrahydronapthylenyl,1,2,3,4-tetrahydrophenanthrenyl, tetrahydroanthracenyl,1,4-dihydro-1,4-methanonaphthalenyl, and benzodioxolyl.

The term “heteroaryl” refers to a heteroaromatic (heteroaryl) grouphaving one, two, or three aromatic rings containing from 1-4 heteroatoms(such as nitrogen, sulfur, or oxygen) in the aromatic ring. Heteroarylgroups include one, two, or three aromatic rings structures containingfrom 1-4 heteroatoms fused with one or more additional nonaromatic ringshaving from 4-9 members. Heteroaryl groups containing a single type ofheteroatom in the aromatic ring are denoted by the type of heteroatomthey contain, thus, nitrogen-containing heteroaryl, oxygen-containingheteroaryl and sulfur-containing heteroaryl denote heteroaromatic groupscontaining one or more nitrogen, oxygen or sulfur atoms respectively.Exemplary heteroaryl groups include, without limitation, pyridyl,pyrimidinyl, triazyl, quinolyl, quinazolinyl, thiazolyl,benzo[b]thiophenyl, furanyl, imidazolyl, indolyl, and the like.

The term “carboxyl containing polymer” refers to a polymer containingmonomer units having carboxyl groups present as the free acid or as acorresponding salt, or carboxylic acid groups present in an activatedform such as an ortho-nitrophenyl ester or an anhydride. Where themonomers contain carboxylic acids as an anhydride, the anhydride may beformed between two carboxyl groups within the monomer, resulting in acyclic structure (e.g., maleic anhydride monomer units found in SMApolymers). Carboxyl containing polymers may be derived from a variety ofsources and include both naturally occurring and unnatural carboxylcontaining polymers. Non-limiting exemplary carboxyl containing polymersinclude: PEG-dicarboxylic acid, SMA, carboxyl containing polyacrylicacids or polyacrylonitriles, naturally occurring and unnatural carboxylcontaining polyamides such as PGA, PAA, protein, such as albumin,including for example human serum albumin, polypeptides, naturallyoccurring and unnatural carboxyl containing polysaccharides.

The terms “carbonyl” refers to a —C(═O)— functionality of a ketone oraldehyde.

The term “ketal,” for the purpose of this disclosure, refers to cyclicacetal and cyclic ketal structures formed by the condensation of ahydrated ketone or aldehyde with a 1,2-diol to form 1,3-dioxolane,unless stated otherwise.

The term “carbonyl-containing therapeutic agent” or “carbonyl-containingcompound” refers to therapeutic agents, or more broadly compounds,containing one or more carbonyl groups.

The term “quinone-containing therapeutic agent” or “quinone-containingcompound” refers to a therapeutic agent or more broadly to a compoundbearing one or more aromatic dicarbonyl groups derived from a dihydroxyaromatic compound.

The terms “protected hydroxyl” or “protected amine” refer to hydroxyland amine groups bearing a protective functionality such as Boc or Fmoc.A skilled artisan familiar with standard synthetic methods andprocedures for the preparation of organic molecules may appropriatelyselect suitable protective groups for use in synthesis from the relevantscientific literature or from standard reference textbooks in the fieldsuch as Greene, T. W.; Wuts, P. G. M., Protective Groups in OrganicSynthesis, 3^(rd); John Wiley & Sons: New York, 1999.

The term “linking agent” refers to a molecule that is capable of formingboth a linkage to a polymeric delivery vehicle and a linkage to amolecule that is to be conjugated to the polymeric delivery vehicle,such as a therapeutic agent.

The term “imine-linking agent” refers to a linking agent that may forman imine bond with a carbonyl-containing or quinone-containing moleculethat is to be conjugated to a polymeric delivery vehicle. Imine-linkingagents preferably contain an aromatic amine that may form stable Schiffbases with carbonyl-containing or quinone-containing molecules.

The term “linker conjugated therapeutic agent” refers to a molecule thatcomprises a therapeutic agent that has been covalently bound to alinking agent that is not associated with a polymeric delivery vehicle.

The term “quinol-ester linker conjugated therapeutic agent” refers to amolecule comprising a quinone-containing therapeutic agent in which afirst hydroxyl of the quinol form of the therapeutic agent is covalentlybound to a linking agent by an ester bond (i.e., a quinol-ester), andthe remaining hydroxyl (second hydroxyl) of the quinol is separatelyesterified with a carboxylic acid, where none of the therapeutic agent,the linker, or the carboxylic acid is associated with a polymericdelivery vehicle.

The term “ketal linker conjugated therapeutic agent” refers to amolecule comprising a quinone-containing therapeutic agent or acarbonyl-containing therapeutic agent that has been covalently bound toa linking agent by formation of a ketal linkage between a 1,2-diol(vicinal diol) of a linker and one carbonyl of a quinone-containingtherapeutic agent, or a carbonyl of carbonyl-containing therapeuticagent, where neither the therapeutic agent, nor the linker areassociated with a polymer delivery vehicle.

The terms “polymer-modified therapeutic agent” and “polymer conjugate”refer to compositions in which a therapeutic agent is covalentlyattached to a polymeric vehicle via a linking agent. For the purposes ofthis application, such compositions are typically formed by reacting alinker conjugated therapeutic agent with a polymeric vehicle underconditions that will result in the formation of a bond between thelinker of the linker conjugated therapeutic agent and the polymericvehicle.

While the term “PGA” refers generally to poly(glutamic acid), unlessstated otherwise, for the purposes of this invention the term “PGA”refers to poly-L-glutamic acid), alternatively indicated aspoly(L-glutamic acid). Similarly, while the term “PAA” refers generallyto poly(aspartic acid), unless stated otherwise, for the purposes ofthis invention the term “PAA” refers to poly-L-aspartic acid),alternatively indicated as poly(L-aspartic acid).

For the purpose of this invention, where the molecular weight of apolymer is given, it is to be understood that the molecular weightrepresents the average molecular weight of the subject polymer, unlessit is stated otherwise. Where a range of polymer molecular weight isgiven, an individual molecular weight value within the range representsan average molecular weight of the subject polymer. Where the molecularweight of a “polymer conjugate” is given, the molecular weightrepresents that of the sodium salt unless stated otherwise.

B. POLYMERIC VEHICLES FOR THE DELIVERY OF POLYMER-MODIFIEDQUINONE-CONTAINING OR CARBONYL-CONTAINING THERAPEUTIC AGENTS

1. Suitable Polymeric Vehicles for the Delivery of Carbonyl-Containingand Quinone-Containing Therapeutic Agents.

A variety of polymers are useful as polymeric delivery vehicles fordelivering therapeutic agents to tumor cells or tumor tissues. Amongthese polymers are carboxyl-containing polymers including, but notlimited to, poly(styrene-co-maleic anhydride) copolymers (SMA),N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with carboxylcontaining peptides. Other suitable polymeric delivery vehicles includecarboxyl-containing polyamides such as polyglutamic and polyasparticacids, and modified polyethylene glycols (PEG). Association oftherapeutic agents with polymers provides for a larger molecule, whichmay be selectively accumulated in tumor tissues via the EPR effect.Preferred polymers of the invention bearing free carboxylic acidmoieties, which are suitable for the attachment of quinone-containingmolecules, such as β-lapachone, through the linking groups are describedbelow.

2. SMA and HPMA Polymers:

Particularly suitable polyvinyl polymers for the delivery ofcarbonyl-containing or quinone-containing therapeutic agents, such asβ-lapachone, include those bearing carboxyl moieties that may serve assites for the conjugation of linkers suitable for attaching thetherapeutic agents to polymers. One such type of polyvinyl polymer ispoly(styrene-co-maleic anhydride) polymers (“SMA polymers” or “SMA”). Aparticular advantage of SMA is its ability to non-covalently bond toalbumin in vivo, which further aids the observed EPR effect. Inaddition, the amphiphilic nature of SMA enables a range of formulationpossibilities including as an aqueous formulation for parenteraladministration (e.g., intravenous, intramuscular, intraperitoneal, andsubcutaneous injection).

In certain embodiments, the SMA preferably comprises the following mainchain units:

in desired ratios. Desired ratios for styrene to maleic anhydrideresidues (expressed as moles of styrene residues to moles of maleicanhydride) vary for different molecular weight SMA polymers. For lowermolecular weight SMA polymers desired ratios are from about 1.6:1.0 toabout 1:1. Other suitable ratios include about 1.4:1 and about 1.3:1.For higher molecular weight SMA polymers the ratio of styrene to maleicanhydride residues increases to about 3:1 at 9,500 Da, and about 4:1 at11,000 Da. The upper limit of maleic anhydride residues in SMA polymersis generally not greater than 50 mol %, with the upper limit being about60 mol %. Preferred SMA moieties comprise styrene-co-maleic anhydrideunits of the form:

where “g” and “h” represent integer values consistent with those ratiosof styrene residues to maleic anhydride residues described above.

In other embodiments, SMA polymers may also contain maleic acid halfester residues of the form

In the above maleic acid half ester residue, R is: (1) a linear loweralkyl residue having 1-5 carbon atoms, such as methyl, ethyl, propyl,butyl, pentyl, etc., preferably normal alkyl groups, more particularlyn-butyl; (2) a monohydroxy-alkyl ether residue of di- or trihydricalcohols, for example: alkylene glycol mono-lower alkyl ether residue,wherein the alkylene group is ethylene or butylene and the lower alkylgroup has 1-4 carbon atoms and is preferably ethyl or n-butyl, moreparticularly n-butyl; (3) a polyethyleneglycol mono-alkyl ether residuerepresented by the formula —(C₂H₄O)_(k)—R′ wherein R′ is a linear alkylgroup having 1-8 carbon atoms, and “k” is from 2 to 20; or (4) aglycerin dialkyl ether residue, wherein the alkyl groups have 1-4 carbonatoms. The term “half ester” used herein means that one of the twocarboxyl groups of maleic acid formed by opening the ring of maleicanhydride has been esterified. It is preferable that substantially allmaleic acid residues, other than maleic anhydride residues, are halfesterified but a part thereof may be free maleic acid residues.

SMA polymers useful for forming conjugates may have an average molecularweight of about 1,000-20,000 Da, as determined by gel permeationchromatography. Other suitable molecular weight ranges for SMA polymersinclude from about 1,000 to about 5,000 Da, or from 1,100 to about 4,000Da or from about 1,200 to about 3,000 Da or from about 1,300 to about2,000 Da. Additionally, SMA polymers useful for forming conjugates mayhave an average molecular weight from about 8,000 Da to about 17,000 Daor from about 10,000 Da to about 15,000 Da or from about 11,000 to about13,000 Da. Particularly useful SMA polymers for forming conjugates mayhave an average molecular weight of about 1,200 Da, about 1,600 Da orabout 12,000 Da. All molecular weights recited are average molecularweights of the polymers prior to their conjugation withcarbonyl-containing or quinone-containing therapeutics agents unlessstated otherwise. SMA polymers may be prepared by methods known in theart, including half-esterifying styrene-maleic anhydride copolymers withmono-alcohols, or may be purchased from a variety of sources includingSartomer (Exton, Pa.) and Sigma-Aldrich (St. Louis, Mo.).

3. N-(2-hydroxypropyl)methacrylamide Copolymers (HPMA Copolymers)

In addition to those polymers already discussed, polymers ofN-(2-hydroxypropyl)methacrylamide may also be employed in thepreparation of polymer-modified carbonyl-containing orquinone-containing therapeutic agents. Suitable HPMA copolymers includethose bearing carboxyl groups, protected carboxyl groups or activatedcarboxyl groups that are capable of reacting with the hydroxyl or aminefunctionalities of linkers of formulas I, II or III. In a preferredembodiment, the HPMA copolymers are copolymers ofN-(2-hydroxypropyl)methacrylamide and N-methacryloylamino acids, orN-methacryloylpeptides (particularly those peptides where theN-methacryloyl group is on the N-terminal amine). Free carboxyl groupsof both N-methacryloylamino acids, or N-methacryloylpeptides areprotected as required for the polymerization withN-(2-hydroxypropyl)methacrylamide to form the desired HPMA copolymers. Avariety of readily hydrolyzable protecting groups may be employed toprotect the terminal carboxyl group of N-methacryloylamino acids orN-methacryloylpeptides such as methyl, ethyl, or benzyl esters,particularly preferred are aryl esters such as 4-nitrophenyl estersgroups. Protecting groups such as 4-nitrophenyl esters are particularlyadvantageous as they may be directly reacted with the amine or hydroxylfunctionalities present in the linkers of linker conjugated therapeuticagents described below to form polymer-modified carbonyl-containing orquinone-containing therapeutic agents having an ester or amide bond tothe peptide or amino acid and of the HPMA copolymer. Reaction ofnitrophenyl esters with linker conjugated therapeutic agents to formamide or ester linkages may be conducted in polar aprotic solvents suchas DMF, DMSO or acetonitrile at elevated temperatures. Alternatively,N-methacryloylamino acids, or N-methacryloylpeptides bearing carboxylprotecting groups, including 4-nitrophenyl esters, may be deprotected toyield the corresponding free acids, which may be coupled to amine orhydroxyl groups present in the linkers of linker conjugated therapeuticagents in using suitable dehydrating agents such asdicyclohexylcarbodiimide (DCC).

The preparation of monomers for preparing HPMA copolymers may beconducted by methods known in the art. More specifically,N-(2-hydroxypropyl)methacrylamide may be prepared by methods such asthose described by Ulbirch, K., et al. in J. Controlled Release, 64: 63(2000) and N-methacryloylamino acids, or N-methacryloylpeptides may alsobe prepared by Schotten-Baumann reactions employing N-methacryloylchloride in aqueous alkaline medium as described by Etrych et al. inSynthesis of HPMA Copolymer Containing Doxorubicin Bound via a HydrazoneLinkage. Effect of Spacer on Drug Release and in vitro Cytotoxicity,Macromolecular Bioscience 2: 43-52 (2002). Any suitably protectedN-methacryloylamino acid or N-methacryloylpeptide may be employed in thepreparation of HPMA copolymers. The synthesis of other N-methacryloyl4-nitrophenyl peptide esters suitable as monomers for the preparation ofHPMA copolymers are described in WO 03/053473. Such peptides includeGlyPheGly, GlyPheLeuGly, and GlyLeuPheGly. Some suitableN-methacryloylamino acid, and N-methacryloylpeptide 4-nitrophenyl estersinclude: N-methacryloylglycine 4-nitrophenyl ester,N-methacryloylalanine 4-nitrophenyl ester, N-methacryloyl-β-alanine,6-(2-methyl-acryloylamino)-hexanoic acid 4-nitro-phenyl ester (i.e.,N-methacryloyl(6-aminohexanoic acid) 4-nitrophenyl ester),4-(2-methyl-acryloylamino)-benzoic acid, N-methacryloylglycylglycine4-nitrophenyl ester, N-methacryloylglycyl-L-leucylglycine 4-nitrophenylester, N-methacryloylglycyl-DL-phenylalanyl-L-leucylglycine4-nitrophenyl ester.

Random copolymers of N-(2-hydroxypropyl)methacrylamide with one or moreN-methacryloylamino acids, or one or more N-methacryloylpeptides (e.g.,4-nitrophenyl esters of N-methacryloylamino acids, orN-methacryloylpeptides) may be prepared by methods described in the artsuch as those in Etrych et al in Synthesis of HPMA Copolymer ContainingDoxorubicin Bound via a Hydrazone Linkage. Effect of Spacer on DrugRelease and in vitro Cytotoxicity, Macromolecular Bioscience 2: 43-52(2002). The reaction is typically conducted by free radicalprecipitation polymerization in acetone using AIBN (0.4-0.7% based uponthe weight of monomers employed) as a free radical catalyst. Typicalranges for N-(2-hydroxypropyl)methacrylamide monomers toN-methacryloylamino acid, or one or more N-methacryloylpeptide monomersare from about 25:1 to about 10:1. The reactions are typically carriedout at 50° C. for 24 hours. Polymers suitable for the formation ofconjugates typically contain from about 30 monomers (about 4,000 Da) toabout 3,000 (about 400,000 Da), where the estimated molecular weightsare based upon the calculated molecular weight ofN-(2-hydroxypropyl)methacrylamide and will be larger based upon theamino acids or peptides used in the copolymerization.

4. Polyamides

Polyamides suitable for the delivery of carbonyl-containing orquinone-containing therapeutic agents, such as β-lapachone, includethose bearing free carboxyl functionalities that may serve as sites forthe conjugation of linkers suitable for attaching the therapeutic agentsto the polymers via the quinone functionality. Preferred polyamidesinclude, but are not limited to PGA polymers (poly(L-glutamic acid),poly(D-glutamic acid), and poly(DL-glutamic acid)), PAA polymers(poly(L-aspartic acid), poly(D-aspartic acid), and poly(DL-asparticacid)), and copolymers comprising one or more isomer of aspartic acidwith one or more isomer of glutamic acid. More preferred polyamidepolymers include poly(L-aspartic acid) and poly(L-glutamic acid). Mostpreferred is poly(L-glutamic acid). Unless stated otherwise, PGA and PAAare understood to refer to (poly(L-glutamic acid) and (poly(L-asparticacid) respectively.

Polyglutamic acid (PGA) sodium salt and the free acid have the form:

where “n” represents an integer value consistent with the molecularweight range of polyglutamic acid polymers suitable for the preparationof polymer conjugates.

The polyglutamic acids and polyaspartic acids for the preparation ofconjugates of the invention may have an average molecular weight ofabout 5,000 Da to about 100,000 Da, or from about 7,000 Da to about80,000 Da, or from about 10,000 Da to about 70,000 Da. Other suitablemolecular weight ranges for polyglutamic acids and polyaspartic acidsare from about 12,000 Da to about 28,000 Da or from about 15,000 Da toabout 24,000 Da, or from about 20,000 to about 80,000 Da, or from about30,000 to about 70,000 Da, or from about 45,000 to about 60,000 Da.Particularly useful polyglutamic acids and polyaspartic acids forforming conjugates may have an average molecular weight of about 17,000Da, or an average molecular weight of about 22,000 Da, or an averagemolecular weight of about 56,000 Da may also be employed. All molecularweights given are average molecular weights of the sodium salt of thepolymers prior to their conjugation with carbonyl-containing orquinone-containing therapeutics agents unless stated otherwise.Polyglutamic acid and polyaspartic acid molecular weights may bedetermined by multi-angle laser light scattering (MALLS), viscosity,and/or gel permeation chromatography.

Polyglutamic and polyaspartic acid polymers, including copolymers, maybe made by methods known in the art including the condensation ofmonomers in the presence of suitable catalysts (e.g., acids or bases) ordehydrating agents, such as N,N′-dicyclohexylcarbodiimide.Alternatively, polyaspartic acid and polyglutamic acid are availablefrom commercial sources (e.g., Sigma-Aldrich, St. Louis, Mo.).

3. Polyethylene Glycols

Polyethylene glycols (H[OCH₂CH₂]_(d)OH, where d represents the number ofrepeating units found in the PEG molecule) suitable for the delivery ofcarbonyl-containing or quinone-containing therapeutic agents, such asγ-lapachone, include those modified to bear free carboxylfunctionalities that may serve as sites for attachment of linkerconjugated therapeutic agents via the linker moiety. Polyethyleneglycols having different average molecular weights are available from avariety of different suppliers, including Sigma-Aldrich (St. Louis, Mo.)For the purpose of preparing polymer-modified carbonyl-containing orquinone-containing therapeutic agents, PEG having a molecular weightrange of about 20,000 to about 60,000 Da is preferred, although othermolecular weight ranges may be employed, including from about 1,000 toabout 10,000 Da, about 10,000 Da to about 20,000 Da, and about 60,000 toabout 80,000 Da.

Modification of PEG molecules to incorporate moieties bearing freecarboxyl functionalities may be accomplished by a variety of means,including the reaction of PEG with halogenated-carboxylic acids in thepresence of a base. For the purpose of preparing the PEG derivative thecarboxyl group is generally protected as an ester (e.g., methyl, ethyletc.) that may later be cleaved under suitable conditions. One suitablegroup of halogenated-carboxylic acid esters that may be employed ishalogenated aliphatic carboxylic acid esters. Preferably, thehalogenated aliphatic acid esters are ω-halogenated aliphatic carboxylicacid or ω-halogenated alkyl carboxylic acid of the formhalogen-(CH₂)_(c)C(═O)—O-protecting group, where “c” is an integer from1-12, which upon reaction with PEG and deprotection give rise to aPEG-dicarboxylic acid of the form:HOOC(CH₂)_(c)O[OCH₂CH₂]_(d)O(CH₂)_(c)COOH, where “d” is an integer valueconsistent with molecular weight of PEG that may be employed to formpolymer conjugates. PEG-dicarboxylic acid may be represented asHOOC—CH₂—O-PEG-O—CH₂—COOH, which is shown for the di-acetic acid. Theaverage molecular weight of the PEG molecule may be give by a subscriptto the PEG repeating unit (e.g., HOOC—CH₂—O-PEG_(40kDa)-O—CH₂—COOH. fora 40,000 Da PEG). A particularly useful halogenated aliphatic carboxylicacid ester for preparing the above-described dicarboxylic acid forms ofPEG is ethyl bromoacetate.

Reaction of PEG with esters of halogenated carboxylic acids may beconducted by procedures described in the art, such as those described byGreenwald R. B. et al., Drug Delivery Systems Water Soluble Taxol2′-Poly(ethylene glycol) Ester Prodrugs-Design and in VivoEffectiveness, J. Med. Chem. 39: 424-31 (1996); Veronese M. et al.,Physico-Chemical and Pharmacokinetic Characterization of MonethoxyPoly(Ethylene Glycol)-Derivatized Superoxide Dismutase, J. ControlledRelease, 10, 145-54 (1989); or Gebhardt, H. et al. Soluble Polymers inOrganic Chemistry 5. Preparation of Carboxyl- and Amino-TerminalPolyethylene Glycols of Lower Molecular Weight, Polymer Bull. 18: 487-93(1987). Generally, the reactions of PEG with esters of halogenatedcarboxylic acids are conducted in the presence of a suitable base (e.g.,K⁺ tert-butoxide⁻). The carboxyl groups are then de-esterified inaqueous NaOH, and the free acid recovered after acidification with asuitable acid (e.g., aqueous HCl).

C. CARBONYL AND QUINONE CONTAINING THERAPEUTIC AGENTS

The compositions and methods of delivering therapeutic agents describedherein are generally applicable to therapeutic agents bearing carbonylor quinone functionalities. Exemplary carbonyl and quinone-containingtherapeutic agents include, but are not limited to, lobeline,acebutolol, methyprylon, haloperidol, molindone, naloxone, oxycodone,methadone, ketanserin, tolmetin, ketoprofen, nabumetone, canrenone,canrenonate, mebendazole, oxolinic acid, tetracycline,chlortetracycline, oxytetracycline, demeclocycline, doxycycline,minocycline, daullorubicin, doxorubicin, mitoxantrone, plicamycin,mitomycin, indan-1,3-dione, anisindione, testosterone (and related C-17esters, e.g., propionate, enanthate, cypionate), dihydrotesterone,cyproterone acetate, estrone, progesterone, medroxyprogesterone acetate,hydroxyprogesterone caproate, norethindrone, norethynodrel, megestrolacetate, norgestrel, mifepristone, methandrostenolone, oxandrolone,testolactone, cyproterone acetate, prednisone, prednisolone,betamethasone, dexamethasone, other 3-, 17-, or 20-ketosteroids (e.g.,dehydroepiandorsterone, androstenedione, cortisol, cortisone,aldosterone, etc.), and in a preferred embodiment, β-lapachonecompounds. Where the carbonyl groups of a quinone-containing therapeuticagent are not equivalent, both of the individual regioisomers, andmixtures of both regioisomers, that may be formed by attachment of thelinking agent to the non-equivalent carbonyls of the quinone, areconsidered within the scope of this invention. Similarly,polymer-modified forms of each of the individual regioisomers that maybe formed from asymmetric quinones, and mixtures containingpolymer-modified forms of both regioisomers are considered within thescope of this invention.

The β-lapachone compounds of the invention include β-lapachone andanalogs thereof. As discussed above, β-lapachone has the followingchemical structure:

β-lapachone analogs include compounds that are structural derivatives ofβ-lapachone, differing from β-lapachone by substitution of one, two,three, four, or more elements of βlapachone with a different group orelement. For example, a hydrogen at the 3 or 4 position may besubstituted with a hydroxy or a C₁-C₄ alkyl, wherein the C₁-C₄ alkyl isoptionally substituted with a hydroxy. Preferred substituents include3-hydroxy and 3-methanolyl. Further, each of the methyl groups atposition 2 may be independently substituted with a hydrogen. Theβ-lapachone analogs of the invention may also include substitutions ofheteroatoms, for instance, the oxygen at position 1 may be substitutedwith a sulfur atom, and the carbon at position 4 may be substituted withoxygen.

Any β-lapachone analog known in the art may be used as the β-lapachonecompound of the invention. For instance, a number of β-lapachone analogshaving anti-proliferative properties have been disclosed in the art,such as those described in PCT International Application PCT/US93/07878(WO 94/04145), and U.S. Pat. No. 6,245,807, in which a variety ofsubstituents may be attached at positions 3- and 4- on the β-lapachonecompound. PCT International Application PCT/US00/10169 (WO 00/61142),discloses β-lapachone, which may have a variety of substituents at the3-position as well as in place of the methyl groups attached at the2-position. U.S. Pat. Nos. 5,763,625, 5,824,700, and 5,969,163, discloseanalogs with a variety of substituents at the 2-, 3- and 4-positions.Furthermore, a number of journals report β-lapachone analogs withsubstituents at one or more of the following positions: 2-, 3-, 8-and/or 9-positions. See, e.g., Sabba et al., (1984) Journal of MedicinalChemistry 27:990-994 (substituents at the 2-, 8- and 9-positions);(Portela and Stoppani, (1996) Biochem Pharm 51:275-283 (substituents atthe 2- and 9-positions); Goncalves et al., (1998) Molecular andBiochemical Parasitology 1:167-176 (substituents at the 2- and3-positions).

Moreover, structures having sulfur-containing hetero-rings in the “α”and “β” positions of lapachone have been reported (Kurokawa S, (1970)Bulletin of The Chemical Society of Japan 43:1454-1459; Tapia, R A etal., (2000) Heterocycles 53(3):585-598; Tapia, R A et al., (1997)Tetrahedron Letters 38(1):153-154; Chuang, C P et al., (1996)Heterocycles 40(10):2215-2221; Suginome H et al., (1993) Journal of theChemical Society, Chemical Communications 9:807-809; Tonholo J et al.,(1988) Journal of the Brazilian Chemical Society 9(2): 163-169; andKrapcho A P et al., (1990) Journal of Medicinal Chemistry33(9):2651-2655). More particularly, hetero β-lapachone analogs aredisclosed in US Patent Appln. No.: 2004/0266857, entitled “NOVELLAPACHONE COMPOUNDS AND METHODS OF USE THEREOF,” which is hereinincorporated by reference.

Preferred β-lapachone analogs include those in which the carbonyl groupsare the vicinal diones at the 5 and 6 positions of the3,4-dihydro-2H-benzo[h]chromene-5,6-dione nucleus.

D. POLYMER CONJUGATION

1. Linking Functionalities for Carbonyl-Containing andQuinone-Containing Molecules.

Therapeutic agents containing a quinone or carbonyl functionality mayadvantageously be conjugated to the above-described carboxyl-containingpolymers of this invention by several linking groups including imines(Schiff's bases), quinol-esters, and ketal linkages. Where thetherapeutic agent contains a quinone functionality that is employed inthe formation of the polymer conjugate, the quinone may be a 1,2-quinoneor a 1,4-quinone even though the 1,2-quinones are depicted.

In accordance with the present invention, one embodiment encompassespolymer-modified quinone-containing or carbonyl-containing therapeuticagents conjugated to SMA, polyglutamic acid, or poly aspartic acids byan imine, quinol-ester, or ketal linkage. In one embodiment, thetherapeutic agent is β-lapachone and the polymer is SMA or polyglutamicacid.

2. Imine Polymer Conjugates.

Linking agents capable of forming an imine bond between an amine of thelinking agent and a carbonyl or quinone functionality of a therapeuticagent may be employed to form conjugates with polymer vehicles. Linkingagent amines for the formation of imine bonds are advantageouslyaromatic amines that form stable Shiff bases. In addition to providingan amine group for formation of an imine linkage to the therapeuticagent, imine-linking agents also provide a second amine for conjugatingthe linking agent to a carboxyl group on a polymeric vehicle via amidelinkage. Linking agents suitable for conjugation of carbonyl-containingor quinone-containing compounds are generally of the form of formula(I):

where

M is a spacer group selected from the group consisting of: —(C₁-C₈)alkyl-, —(CH₂)_(q)—O—(CH₂)_(r)—, —C(═O)—O—(CH₂)_(r)—,—(C₃-C₇)cycloalkyl-, -aryl-C(═O)—O—(CH₂)_(r)—, —C(═O)—O-aryl-(CH₂)_(r)—,-heteroaryl-C(═O)—O—(CH₂)_(r)—, and —C(═O)—O-heteroaryl-(CH₂)_(r)—;

Z is —OH, a protected amine, or a protected hydroxyl;

each R₁ is independently selected from the group consisting of:hydrogen, halogen, and —(C₁-C₄) alkyl;

q is from 0-6;

r is from 2-6; and

t is from 0-4.

Linking agents of formula I may be prepared by a variety of syntheticroutes or purchased commercially. For example: (4-aminophenyl)methanol;1-(4-aminophenyl)ethanol; 4-(aminomethyl)aniline;2-(4-aminophenyl)ethanol; 4-(2-aminoethyl)aniline; 2-(diethylamino)ethyl4-aminobenzoate hydrochloride; 2-(diethylamino)ethyl4-amino-2-chlorobenzoate hydrochloride; 4-[(5-phenoxypentyl)oxy]anilineare commercially available from Sigma-Aldrich (St. Louis, Mo.).

In a preferred embodiment, where linking agents of formula (I) are ofthe form

where

Z is a protected amine;

each R₁ is independently selected from the group consisting of hydrogen,halogen, and —(C₁-C₄) alkyl;

r is from 2-6; and

t is from 0-4;

the linking agents may be prepared by reacting 4-nitrobenzoylchloridewith an amine-protected amino alcohol of the form: (protectinggroup)-NH—(CH₂)_(r)—OH, where r is from 2-6. Suitable protecting groupsfor the amino group include, but are not limited to,9-fluorenyl-methoxycarbonyl (Fmoc), 2-chloro-1-indanylmethoxy-carbonyl(Climoc), benz[f]indene-3-methyloxycarbonyl (Bimoc),2,7-di-t-butyl[9-(10,10-dioxo-10,10,-10,10-tetrahydrothioxanthyl)]methyl(DBD-Tmoc), 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc),beta-trimethylsilylethyloxycarbonyl (Teoc), andbis(4′-(nitrophenyl)ethyl carbonyl (Bnpeoc). Fmoc is the preferredprotecting group, and preferred Fmoc protected amino alcohols are of theform Fmoc-NH—(CH₂)_(r)—OH, where r is from 2-6. The reaction istypically conducted in methylene chloride at room temperature for 1hour. Following completion, the reaction is washed with water andsaturated aqueous solution of sodium chloride. The methylene chloride isdried with Na₂SO₄, and concentrated to yield the 4-nitro-benzoic acidester. The nitrogen functionality is subsequently reduced by catalytichydrogenation in the presence of a noble metal catalyst, such aspalladium on charcoal. The reaction is typically conducted at roomtemperature under 1 atmosphere of nitrogen for 2 hours. Followingreduction the catalyst is removed by filtration and the linker offormula (I) is purified by silica gel chromatography as necessary.

In a more preferred embodiment, where t is 0, r is 2, and Z isFmoc-protected amine, the imine linking agent is2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl 4-aminobenzoate.Formation of linkers of formula (I) is exemplified by the preparation of2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl 4-aminobenzoate inExamples 1 and 2, and Scheme 1.

Linkers of formula (I) and carbonyl-containing or quinone-containingtherapeutic agents may be conjugated via the aromatic amine of thelinker to form a linker conjugated therapeutic agent in a condensationreaction. In the reaction, the aromatic amine of the linking agent and acarbonyl of the therapeutic agents are converted to an imine linkage.The condensation reaction may be conducted in the presence of anysuitable agents including, but not limited to, TiCl₄, p-toluene sulfonicacid, Si(OEt)₄, sulfuric acid, ZnCl₂, ZnBr₂, and ionic liquids (forexample 1-alkyl-2,3-dimethylimidazolium, 1-alkyl-3-methylimidazolium,1-alkylpyridinium, 1-alkyl-2,3-dimethylimidazolium,1-alkyl-3-methylimidazolium, 1-alkylpyridinium,1-alkyl-3-methylimidazolium, 1-alkylpyridinium,1-alkyl-3-methylimidazolium, 1-alkylpyridinium,1-alkyl-3-methylimidazolium, 1-alkyl-3-methylimidazolium,1-alkylpyridinium, 1-alkyl-3-methylimidazolium, 1-alkylpyridinium, and1-alkyl-2,3-dimethylimidazolium produced by Acros Organics, availablethrough Fisher Scientific International, Hampton N.H., or FisherChemicals, Fairlawn, N.J.), The preferred condensing agent is TiCl₄.Typically the condensation reaction is conducted employing a slightmolar excess of the linking agent over the carbonyl-containing orquinone-containing therapeutic agent in methylene chloride at roomtemperature in the presence of triethylamine. The conjugation of linkersof formula (I) to carbonyl-containing or quinone-containing therapeuticagents is exemplified by the formation of2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl4-{[(6Z)-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]chromen-6(5H)-ylidene]amino}benzoatein Example 3 and Scheme 1.

Formation of a polymer-modified therapeutic agent may be conducted byreacting the Z group hydroxyl or amine of the linker conjugatedtherapeutic agent with a carboxyl-containing polymer to form therespective ester or amide bond. The reaction is commenced by firstdeprotecting the Z group of the linker to yield a deprotected amine orhydroxyl group, and condensing the hydroxyl or amine with a polymercarboxyl group. Suitable conditions for the condensation of thedeprotected amine or hydroxyl groups with the polymer carboxyl groupinclude reaction in a polar aprotic solvent such as DMF in the presenceof a nucleophilic catalyst such as dimethyl amino pyridine and adehydrating agent including, but not limited to, DCC(dicyclohexylcarbodiimide), HBTU(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), HATU(N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate), BOP((benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate), EDC(N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide), DMC(2-chloro-1,3-dimethylimidazolinium chloride), carbonyldiimidizole andother standard peptide coupling agents. HBTU is generally preferred.Alternatively, where the polymer contains acid anhydride groups, as inthe case of styrene maleic anhydride polymers, the deprotected Z grouphydroxyl or amine functionalities may be reacted with the polymerdirectly to form polymer-modified therapeutic agents having an ester oramide bond to the polymer in a mixture of aqueous NaHCO₃ and THF.

While it is possible to separately deprotect the Z group amines and forman amide linkage with a carboxyl-containing polymers, where the amineprotecting group is Fmoc, or an Fmoc-related protecting group such as2-chloro-1-indanylmethoxy-carbonyl (Climoc),benz[f]indene-3-methyloxycarbonyl (Bimoc),2,7-di-t-butyl[9-(10,10-dioxo-10,10,-10,10-tetrahydrothioxanthyl)]methyl(DBD-Tmoc), 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc),beta-trimethylsilylethyloxycarbonyl (Teoc) or (Bnpeoc), the formation ofa polymer-modified therapeutic agent is preferentially performed withoutseparately deprotecting and isolating the free amine product. Formationof polymer-modified therapeutic agents from linker conjugatedtherapeutic agents protected with Fmoc or Fmoc-related protecting groupsmay be conducted without isolation of the deprotected conjugate byreacting the linker conjugated therapeutic agent with1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) at room temperature indimethylformamide (DMF). A carboxyl-containing polymer, such as PGA,PAA, or SMA containing maleic acid half ester residues, is then addedfollowed by triethylamine and a condensing agent (preferably HBTU).After reacting at room temperature for 8 to 16 hours, the reactionmixture is mixed with aqueous sodium bicarbonate, filtered and thesolution freeze-dried. The resulting solid is dialyzed and the productisolated by freeze-drying. This process is exemplified by the formationofN-{2-[(4-{[(6Z)-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]-chromen-6(5H)-ylidene]amino}benzoyl)oxy]ethyl}γ-poly-L-glutamatein Example 4 and Scheme 1. Suitable polymers for forming thepolymer-modified therapeutics employing a linking agent of formula Iinclude, but are not limited to, PGA or PAA having a molecular weightfrom about 5,000 Da to about 100,000 Da, or from about 7,000 Da to about80,000 Da, or from about 10,000 Da to about 70,000 Da. Other suitablemolecular weight ranges for PGA and PAA polymers for forming thepolymer-modified therapeutics employing a linking agent of formula Iinclude, but are not limited to, from about 12,000 Da to about 28,000Da, or from about 15,000 Da to about 24,000 Da, or from about 20,000 toabout 80,000 Da, or from about 30,000 to about 70,000 Da, or from about45,000 to about 60,000 Da. Particularly useful PGA and PAA polymers forforming polymer-modified therapeutics may have an average molecularweight of about 17,000 Da, or an average molecular weight of about22,000 Da, or an average molecular weight of about 56,000 Da, and PGA ispreferred.

In a preferred embodiment, where the therapeutic agent is β-lapachone,the imine polymer conjugate is of the form

where r is from 1 to 6. In a more preferred embodiment, r is 2, thepolymer is polyglutamic acid, and the β-lapachone conjugate is of theform

where polymer indicates the points of attachment to other glutamic acidresidues of the polyglutamic. While a single regioisomer is indictedabove, the opposite regioisomer, which involves imine bond formation atthe other quinone carbonyl may be prepared and is considered within thescope of this invention.

The quantity of therapeutic agents bound to carboxyl-containing polymersvia a linking agent of formula (I) may be determined by hydrolysis ofthe imine linkage and quantitation of the released therapeutic agent.Hydrolysis of the imine may be conducted in aqueous acid (1.0 M HCl),and the reaction may be warmed as necessary to complete the hydrolysis.Free therapeutic agents may be quantitated by any suitable meansincluding HPLC or UV/Vis spectroscopy. A polymer conjugate may containtherapeutic agents from about 2% to about 35% by weight based upon thepolymer conjugate. In other embodiments, a polymer conjugate may be fromabout 6% to about 30% of the therapeutic by weight, or about 7% to about28% of the therapeutic by weight, or about 10% to about 20% of thetherapeutic by weight. For polymer-modified β-lapachone compounds wherethe polymer is polyglutamic acid, conjugates may be from about 5% toabout 40% by weight of β-lapachone, in other embodiments conjugates arefrom about 8% to about 25% β-lapachone by weight, or about 10% to about20% β-lapachone by weight based upon the total weight of thepolyglutamic acid modified β-lapachone composition.

3. Quinol-Ester Polymer Conjugates.

Quinone-containing therapeutic agents may be conjugated to polymericvehicles through an ester bond between a linking agent and one hydroxylof a reduced quinone (i.e., a quinol) functionality, where the secondquinol hydroxyl group is separately esterified with a carboxylic acid.Quinol-ester polymer conjugates are of the generic form

which is shown for a 1,2 quinol, although quinol-ester polymerconjugates may also be formed with 1,4 quinols (quinones). Where thecarbonyl groups of a quinone are not equivalent, both individualregioisomers, and mixtures of both regioisomers, that may be formed byattachment of the linking agent to the non-equivalent carbonyls of thequinone, are considered within the scope of this invention. Linkingagents suitable for forming ester-conjugated quinols include a group,such as an amino group, capable of attaching the linking agent to acarboxyl group on a polymeric vehicle by an amide linkage.

Carboxylic acids suitable for separately esterifying the second quinolhydroxyl are of the formula R₂COOH, where R₂ is (C₁-C₈) alkyl,cycloalkyl, aryl, heterocycle, heteroaryl, aryl-alkyl, or alkylaryl,wherein said alkyl and cycloalkyl groups within R₂ may be substitutedwith one or more substituents independently selected from the groupconsisting of —H and —F, and wherein said aryl groups within R₂ may besubstituted with one or more substituents independently selected fromthe group consisting of —H and halogen. In some preferred embodiments,R₂ is (C₁-C₈) alkyl, in other preferred embodiments R₂ is (C1-C2) alkyl,and in other preferred embodiments R₂ is methyl.

In one embodiment the linking agents include natural and unnatural aminoacids of the formula (II):

where

each R₃ and R₄ are independently selected from the group consisting ofhydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄) alkyl-aryl, aryl,and heteroaryl;

R₅ is selected from the group consisting of hydrogen, —(C₁-C₈) alkyl,—(C₁-C₈) fluoroalkyl, aryl, and heteroaryl;

R₆ is selected from the group consisting of -tert-butoxycarbonyl andCBZ; and

m is from 1 to 8;

alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle.

Linkers of formula II where m is 1 and R₄ and R₅ taken together with thecarbon and nitrogen atoms bearing them form a 4 to 7 memberednitrogen-containing heterocyclic or heteroaryl ring are of the form:

where the dashed line represents the remainder of the 4-7 memberednitrogen containing heterocyclic or heteroaromatic ring. In one suchembodiment, the linker of formula II is proline, or more preferablyL-proline.

In some embodiments, m is from 1-3, and in other embodiments m is from4-8. In other embodiments, at least one independently selected R₃ or Rgroup is not H. In a more preferred embodiment, R₃ and R₄ are H, and mis from 1-6. In still another preferred embodiment, R₃ and R₄ are H, mis 1, R₅ is hydrogen and R₆ is tert-butoxycarbonyl; and the linkingagent is N-tert-butoxycarbonyl glycine (tBoc glycine or Boc glycine):

Linkers of formula (II) may be purchased from a variety of supplierssuch as Sigma-Aldrich. Where R₆ is a t-Boc or CBZ group, the linkers maybe prepared from the amino acid by protection with Boc anhydride(di-t-butyl dicarbonate; di-t-butyl pyrocarbonate; Boc₂O) or CBZchloride (carbobenzyloxy chloride) respectively.

In one embodiment, quinol-ester linker conjugated therapeutic agents areof the form

(which is shown for a 1,2 quinol, although quinol-ester linkerconjugates may also be formed from 1,4 quinones (1,4 quinols));where

R₂ is (C₁-C₈) alkyl, cycloalkyl, aryl, heterocycle, heteroaryl,aryl-alkyl, or alkylaryl, wherein said alkyl and cycloalkyl groupswithin R₂ may be substituted with one or more substituents independentlyselected from the group consisting of —H and —F, and wherein said arylgroups within R₂ may be substituted with one or more substituentsindependently selected from the group consisting of —H and halogen;

each R₃ and R₄ are independently selected from the group consisting ofhydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄) alkyl-aryl, aryl,and heteroaryl;

R₅ is selected from the group consisting of hydrogen, —(C₁-C₈) alkyl,—(C₁-C₈) fluoroalkyl, aryl, and heteroaryl;

R₆ is selected from the group consisting of -tert-butoxycarbonyl andCBZ; and

m is from 1 to 8;

alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle.

In some preferred embodiments R₂ is (C₁-C₄) alkyl, in a more preferredembodiment R₂ is C1 alkyl (i.e., the carboxylic acid of the form R₂COOHis acetic acid).

In one embodiment of quinol-ester polymer conjugates, where the polymeris a carboxyl containing polymer, the conjugates are of the form

where

R₂ is (C₁-C₈) alkyl, cycloalkyl, aryl, heterocycle, heteroaryl,aryl-alkyl, or alkylaryl, wherein said alkyl and cycloalkyl groupswithin R₂ may be substituted with one or more substituents independentlyselected from the group consisting of —H and —F, and wherein said arylgroups within R₂ may be substituted with one or more substituentsindependently selected from the group consisting of —H and halogen;

each R₃ and R₄ are independently selected from the group consisting ofhydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄) alkyl-aryl, aryl,and heteroaryl;

R₅ is selected from the group consisting of hydrogen, —(C₁-C₈) alkyl,—(C₁-C₈) fluoroalkyl, aryl, and heteroaryl; and

m is from 1 to 8;

alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle.

In one preferred embodiment of quinol-ester polymer conjugates, showngenerically for a 1,2-quinol and a carboxyl containing polymer, R₂ is C₁alkyl (i.e., the carboxylic acid of the form R₂COOH is acetic acid), R₃,R₄, and R₅ are each hydrogen, and m is 1:

The preparation of quinol-ester polymer conjugates begins with thepreparation of the linker conjugated therapeutic agent that may beprepared by reacting a linker of formula (II) with a quinone-containingtherapeutic agent and an anhydride of the carboxylic acid R₂COOH in atwo step process. In the first step, a mixture of zinc dust thequinone-containing compound, Na₂S₂O₄, and anN-tert-butoxycarbonyl-protected (t-Boc) linker of formula (IT), adehydrating reagent (such as HBTU) are reacted. The first reaction stepis typically conducted in a polar aprotic solvent, such as DMF at roomtemperature for 12-24 hours, preferably 16 hours. The product of thefirst reaction step is isolated by dilution with an organic solvent(e.g., ethyl acetate), followed by washing of the organic solventreaction mixture with water, drying with Na₂SO₄, and concentration underreduced pressure to yield a residue. In the second step, the residuefrom the first step is dissolved in the acid anhydride of formula(R₅C(═O))₂O, and zinc dust and triethylamine are added. The mixture isheated to 90° C. for 1-4 hours, preferably 2 hours, with stirring. Aftercooling, solvent is removed under reduced pressure and the residue isdissolved in organic solvent (e.g., ethyl acetate), which is washed withwater, dried with Na₂SO₄ and concentrated under reduced pressure toyield the t-Boc protected product quinol-ester linker conjugatedtherapeutic agent. The product is purified by flash columnchromatography on silica gel employing a suitable solvent (e.g., ethylacetate/dichloromethane), and may be further purified bycrystallization. Deprotection of the amine by removal of the t-Boc groupmay be accomplished by any suitable procedure, including reaction withHCl in dioxane to yield the hydrochloride salt. The preparation ofquinol-ester linker conjugated therapeutic agents by this route isexemplified by the preparation of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylN-(tert-butoxycarbonyl)glycinate, which is described in Example 5a andScheme 2a.

In an alternative protocol, preparation of quinol-ester linkerconjugated therapeutic agents may be prepared by reacting a linker offormula (II) with a quinone-containing therapeutic agent in the presenceof Zn dust and HBTU in a first reaction step. The product of the firstreaction step is then treated with Zn dust, triethylamine and theanhydride of the carboxylic acid R₂COOH in a second reaction step. Thefirst reaction step is typically conducted in a polar aprotic solventsuch as DMF at room temperature for 16 hours. The second reaction stepis generally conducted at room temperature for 1-2 hours. Thepreparation of quinol-ester linker conjugated therapeutic agents by thisroute is exemplified by the preparation of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylN-(tert-butoxycarbonyl)glycinate, in Example 5b and Scheme 2b.

The conversion of protected (e.g., Boc protected) quinol-ester linkerconjugated therapeutic agents into their deprotected hydrochloride formis exemplified by the conversion of5-(Acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylN-(tert-butoxycarbonyl)glycinate, prepared as in examples 5a or 5b, toits hydrochloride form(5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylglycinate hydrochloride) as in Examples 6a or 6b.

Formation of quinol-ester polymer conjugates may be conducted byreaction of a suitable carboxyl-containing polymer with the deprotectedquinol-ester linker conjugated therapeutic agent in the presence of adehydrating agent. The reaction is typically conducted in a polaraprotic solvent such as DMF at room temperature for 16 hours. Wherenecessary the time and temperature may be adjusted accordingly; suitabletimes are from 10-30 hours and the reaction may be conducted from about0° to about 50° C. or more preferably from about 15° to about 37° C.After the reaction is complete, the reaction is diluted with water andaqueous HCl is added to precipitate the product quinol-ester polymerconjugated therapeutic agent. The product is collected by centrifugationand is washed with aqueous HCl (e.g., 1.0 N HCl), deionized water, andlyophilized. In preferred embodiments, the carboxyl-containing polymeris polyglutamic acid (PGA) or polyaspartic acid (PAA). PGA and PAAhaving a molecular weight from about 5,000 Da to about 100,000 Da, orfrom about 7,000 Da to about 80,000 Da, or from about 10,000 Da to about70,000 Da may be employed, other molecular weight ranges of PGA and PAAthat may be employed include from about 12,000 Da to about 28,000 Da, orfrom about 15,000 Da to about 24,000 Da, or from about 20,000 to about80,000 Da, or from about 30,000 to about 70,000 Da, or from about 45,000to about 60,000 Da, and particularly PGA of about 17,000 Da, or about22,000 Da, or about 56,000 Da may also be employed. DCC(dicyclohexylcarbodiimide), HBTU(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), HATU(N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate), BOP((benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate), EDC(N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide), DMC(2-chloro-1,3-dimethylimidazolinium chloride), carbonyldiimidizole andother standard peptide coupling agents. The preparation of quinol-esterpolymer conjugates is exemplified by the preparation of thepolymer-modified β-lapachone5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate in Example 7 and the lower portion of Scheme 2a.Alternatively, where the polymer contains acid anhydride groups, as inthe case of styrene maleic anhydride (SMA) polymers, the hydrochloridesalt of the amine may be reacted with the polymer in a mixture ofaqueous NaHCO₃ and THF to form polymer-modified therapeutic agentshaving an amide bond to the polymer. Coupling to an acid anhydridecontaining polymer is exemplified by the reaction of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinatehydrochloride with SMA in Example 8.

The quantity of therapeutic agent conjugated with a carboxyl-containingpolymer via a quinol-ester linkage may be determined by hydrolysis ofthe ester linkage and quantitation of the released therapeutic agent.Hydrolysis of the quinol-ester linkage may be conducted in aqueous base(e.g., 0.01 N NaOH), and the reaction may be warmed as necessary todrive the hydrolysis. Free therapeutic agents may be quantitated by anysuitable means including HPLC, LC-MS (liquid chromatography-massspectroscopy) or UV/Vis spectroscopy. For polymer-modified β-lapachonecompounds linked to PGA or PAA via a quinol-ester linkage, conjugatesare generally from about 5% β-lapachone to about 45% β-lapachone byweight. In some embodiments, conjugates are from about 8% to about 25%β-lapachone or from about 10% to about 20% β-lapachone by weight basedupon the total weight of the polyglutamic acid modified β-lapachone. Inother embodiments, conjugates are from about 25% β-lapachone to about45% β-lapachone by weight, or from about 30% to about 40% β-lapachone byweight based upon the total weight of the polyglutamic acid modifiedβ-lapachone. For polymer-modified β-lapachone compounds linked to SMApolymers via a quinol-ester linkage, conjugates are generally from about5% β-lapachone to about 60% β-lapachone by weight, in some embodiments,they are from about 10% to about 55% β-lapachone or from about 20% toabout 50% β-lapachone, or from about 30% to about 45% by weight basedupon the total weight of the SMA modified β-lapachone.

Although only a single quinol-ester regiosiomer of polymer-modifiedquinone containing compounds, such as polymer-modified β-lapachone, hasbeen shown, both regioisomer of polymer-modified compounds, and mixturesof both regioisomers of polymer-modified compounds are considered to bewithin the scope of this invention. Thus, for example, both quinol esterregioisomers of polymer-modified beta lapachone, where R₂ is C₁ alkyl(i.e., the carboxylic acid of the form R₂COOH is acetic acid), R₃, R₄,and R₅ are each hydrogen, and m is 1:

are considered to be within the scope of this invention, as are mixturesof both of those regioisomers.

4. Ketal Polymer Conjugates.

Therapeutic agents bearing a carbonyl or quinone group may be linked topolymer vehicles through the use of a ketal functionality or linkage.Linking agents suitable for the formation of ketal polymer conugatesprovide vicinal diols for the formation of the ketal functionality orlinkage, and a means, such as an additional hydroxyl or amino group, forattaching the linking agent to a carboxyl group on a polymeric vehiclevia an amide linkage.

In one embodiment, the linking agents are of the form of formula (III):

where

X is a hydroxyl, a protected hydroxyl or a protected amine;

each of R₇ and R₈ is independently selected from the group consisting ofhydrogen, and (C₁-C₄) alkyl;

R₉ is H; and

p is 1-4.

Linking agents of formula (III) may be purchased from a variety ofsources, for example, 1,2,6, hexanetriol, 1,2,4-butanetriol, glycerol,and 3-amino-1,2-propanediol are available from Sigma-Aldrich. In apreferred embodiment the ketal linking agent of formula (III) isglycerol (p is 1 and X is OH). In a related embodiment, the ketallinking agent of formula (III) is glycerol (p is 1 and X is a protectedOH). In other embodiments, the linking agent of formula (III) is notglycerol. In yet another preferred embodiment the linking agent offormula (II) is 3-amino-1,2-propanediol.

Ketal polymer conjugates of carbonyl-containing therapeutic agentslinked by a linking agent of formula (III) to a polymer are of thegeneric form:

Ketal polymer conjugates of quinone-containing therapeutic agents linkedby a linking agent of formula (III) to a polymer are of the genericform:

which is shown for a 1,2-quinone, although 1,4-quinones may also beconjugated via a ketal linkage.

Ketal polymer conjugates are prepared by the formation of the ketallinker conjugated therapeutic agent followed by reaction of the ketallinker conjugated therapeutic agent with a polymeric vehicle. Ketallinker conjugated therapeutic agents may be prepared by reaction of alinker of formula (III) with a quinone-containing therapeutic agent. Thereaction is typically conducted in benzene in the presence of acatalytic amount of acid (e.g., p-toluenesulfonic acid monohydrate) byrefluxing the mixture for 6 hours. After cooling the reaction solvent isremoved and the product is purified by chromatography on silica gel asnecessary. The preparation of ketal linker conjugated therapeutic agentsis exemplified for the conversion of β-lapachone to4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-onein Examples 9a and in Scheme 3 (stereochemistry is not shown).

Where a ketal linked therapeutic agent is prepared employing a linker offormula III where X is a hydroxyl or a protected hydroxyl group, thelinker hydroxyl group may be converted to an amine functionality byconversion to the methansulfonyl ester, followed by reaction with sodiumazide to form the corresponding azidomethyl compound, which may beconverted to an amine by treatment with triphenylphosphine in THF. Thissequence of reaction steps is illustrated in Example 9b for theconversion of4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one(316 mg, 1.0 mmol) to4′-(aminomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one.

Ketal polymer conjugates may be prepared by reacting the ketal linkerconjugated therapeutic agent with a carboxyl-containing polymericvehicle. Where the linker of formula (III) is protected (e.g., X is aprotected amine or hydroxyl group), the protecting group is removed togive a free amine or hydroxyl functionality. The reaction is typicallyconducted in a polar aprotic solvent, such as DMF, in the presence of adehydrating agent for 8 to 16 hours at room temperature. The resultingmixture is diluted with an organic solvent (e.g., chloroform), mixedwith 0.5 M sodium bicarbonate; the resulting aqueous layer is washedagain with organic solvent. After dialyzing the aqueous layer againstdeionized water the product is isolated by lyophilization. In preferredembodiments, the carboxyl-containing polymer is polyglutamic acid (PGA)or polyaspartic acid (PAA). PGA and PAA having a molecular weight fromabout 10,000 Da to about 75,000 Da may be employed, other molecularweight ranges are from about 12,000 Da to about 22,000 Da or from about45,000 to about 60,000 Da, and particularly PGA of about 17,000 Da orabout 56,000 Da may also be employed. Preferred dehydrating agentsinclude, but are not limited to, DCC (dicyclohexylcarbodiimide), HBTU(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), HATU(N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate), BOP((benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate), EDC(N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide), DMC(2-chloro-1,3-dimethylimidazolinium chloride), carbonyldiimidizole andother standard peptide coupling agents. DCC is most preferred.

In a preferred embodiment of ketal polymer conjugates, the ketal linkingagent is glycerol and the polymer is polyglutamic acid. This embodimentis described in Examples 9a and 10, and depicted Scheme 3 for thequinone-containing therapeutic, β-lapachone. In another preferredembodiment the linking agent is 3-amino-propane-1,2-diol. In otherembodiments, ketal polymer conjugates may be prepared with SMA polymershaving an average molecular weight from about 8,000 Da to about 17,000Da or from about 10,000 Da to about 15,000 Da or from about 11,000 toabout 13,000 Da. Other particularly useful SMA polymers for formingketal polymer conjugates may have an average molecular weight of 1,200Da, 16,000 Da or 12,000 Da.

The quantity of therapeutic agent conjugated with a carboxyl-containingpolymer via a ketal linkage may be determined by hydrolysis of the esterlinkage and quantitation of the released therapeutic agent. Hydrolysisof the ketal-linkage, as described in Examples 10 and 11 (c), may beconducted in aqueous base (e.g., NaOH) or acid and the reaction may bewarmed as necessary to drive the hydrolysis. Free therapeutic agents maybe quantitated by any suitable means including LC/MS, HPLC or UV/Visspectroscopy. For polymer-modified β-lapachone compounds linked to PGA(17,000 Da) via a ketal linkage, conjugates may be from about 5%β-lapachone to about 30% β-lapachone by weight, or from about 10% toabout 25% β-lapachone or from about 15% to about 20% β-lapachone basedupon the total weight of the polyglutamic acid modified β-lapachone.

Although only a single ketal polymer conjugate of β-lapachone has beenshown, both ketal polymer conjugate regioisomers of β-lapachonecompounds, and mixtures of both regioisomers, are considered within thescope of this invention.

5. Biological Activity of Polymer Conjugated Therapeutic Agents of theInvention.

Therapeutic agents may be released from their polymer-modified forms bya variety of mechanisms that result in the cleavage of the bonds betweenthe therapeutic agent and the linking agent. The release of thetherapeutic agent may occur from a linking agent while the linking agentis still bound to the polymer, or in the alternative, after the linkerconjugated therapeutic agent is cleaved from the polymer. Release ofquinone-containing and carbonyl-containing therapeutic agents from thepolymer-modified form may occur by a variety of mechanisms including,but not limited to, non-enzymatic hydrolysis of the bond or bondsbetween the therapeutic agent and the linker and enzyme catalyzedcleavage (e.g., esterase or Cytochrome P450 action). The imine,quinol-ester, and ketal linking agents described herein each permitrelease of quinone-containing and carbonyl-containing therapeutic agentsas free quinones and carbonyl compounds without modification of thetherapeutic agent, particularly where the release occurs by hydrolysis.Depending upon the specific therapeutic agent and linker combination,and to some extent the polymer employed in preparing thepolymer-modified therapeutic agent, release of therapeutic agents mayoccur in a variety of locations upon administration to a subject. Wherethe subject is a mammal, release may occur, for example, in theinterstitial or intracellular spaces within a tumor, or within a cell ora cellular organelle such as a lysosome where the polymer conjugatebecomes internalized by cells. Where the polymer-modified therapeuticenters the blood stream, for example when administered as an intravenousformulation, release of therapeutic agents from their polymer-modifiedforms may also occur in plasma during circulation as indicated inExamples 15. Additionally, release of therapeutics from polymer-modifiedforms may occur at a site of administration, such when thepolymer-modified therapeutic is administered intraperitionally orsubcutaneously.

The ability of the polymer-modified therapeutic agents of the inventionto deliver therapeutic agent can be observed in the action of thepolymer-modified therapeutic agent β-lapachone on cells in vitro,described in Example 12, and in nude mice tumor models described inExample 15.

The ability of the polymer-modified therapeutic agents to sustaintherapeutic levels of the agent can be observed in FIGS. 3, 5A and Bcompared with the unmodified therapeutic agent (reference standard).Levels of free therapeutic agent released from the conjugate and total(free plus conjugate) levels of the therapeutic agent are sustained intumor above the levels of the reference (therapeutic agent) startingfrom two or four hours to twenty-four hours post delivery of the agents(FIGS. 3 and 5A). Additionally plasma levels of free and total (freeplus conjugate) levels are sustained above unmodified therapeutic agentlevels for twenty-four hours (FIGS. 3 and 5B).

E. Polymer Conjugated Therapeutic Agents of the Invention

The present invention includes polymer-modified quinone-containing orcarbonyl-containing therapeutic agents. In a preferred embodiment, thetherapeutic agents are conjugated to the polymeric vehicle by an imine,quinol-ester, or ketal linkage formed between the therapeutic agent andthe linking agent. Upon hydrolysis, such linkages advantageously releaseconjugated carbonyl or quinone therapeutic agent from the polymericdelivery vehicle without alteration of the drug. In a more preferredembodiment, the polymeric delivery vehicle selected from PAA or PGA. Inanother more preferred embodiment of the invention, the therapeuticagents are chemotherapeutic agents, such as mitomycin C, doxorubicin,actinomycin D (dactinomycin), β-lapachone or a lapachone analog.

In accordance with the present invention, preferred therapeutic agentsinclude β-lapachone. In preferred embodiments, the polymer-conjugates ofβ-lapachone comprise a polymeric delivery vehicle selected from PGA orPAA bound to β-lapachone by an imine, quinol-ester, or ketal linkageformed between β-lapachone and a linking agent. In some embodiments ofβ-lapachone polymer conjugates, the polymeric vehicle is PGA or PAA, theconjugates may be from about 5% β-lapachone to about 40% β-lapachone byweight, or from about 8% to about 25% β-lapachone, or from about 10% toabout 20% β-lapachone by weight, or from about 26% to about 38%β-lapachone by weight based upon the total weight of the polymerconjugate. Where the polymeric vehicle is SMA, β-lapachone isadvantageously bound to a carboxyl-containing polymeric vehicle by aquinol-ester linkage between the linking agent and β-lapachone. In someembodiments of β-lapachone-SMA polymer conjugates, the conjugates arepreferably from about 25% to about 60% β-lapachone by weight, or fromabout 35% to about 55% β-lapachone by weight, or from about 40% to about50% β-lapachone by weight, based upon the total weight of the polymerconjugate.

The above-described polymer conjugates offer a variety of advantages forthe delivery of therapeutic agents containing a carbonyl or quinonefunctionality. Where effective transport of therapeutic agents, suchβ-lapachone compounds, to tumor cells or tumor tissues is desired topromote therapeutic effectiveness, the polymer conjugates advantageouslypermit selective delivery through the EPR effect in tumor tissues.Furthermore, where therapeutic agents, and particularlychemotherapeutics have limited water solubility, conjugation to watersoluble carboxyl-containing polymers advantageously provides a means ofsolubilizing the therapeutic agent for delivery. Moreover, as thepolymer compositions and linkers of the present invention releasequinone and carbonyl-containing therapeutic agents by hydrolysis of theimine, quinol-ester, or ketal linkages without alteration of thetherapeutic agents, these compositions are advantageously employed todeliver quinone-containing or carbonyl-containing therapeutic agents,without any required the introduction of additional functionalities orgroups that may alter the structure, function, activity or metabolism ofthe therapeutic agents.

F. Methods of the Invention

The methods of the invention include the use of linking agents offormulas (I), (II), or (III) for the preparation of polymer-modifiedcarbonyl-containing or quinone-containing therapeutic agents, in whichthe carbonyl-containing or quinone-containing therapeutic agents arebound to a carboxyl-containing polymer via the linking agents offormulas (I), (II), or (III). More specifically, methods of preparingpolymer conjugates of carbonyl-containing or quinone-containingtherapeutic agents including lobeline, acebutolol, methyprylon,haloperidol, molindone, naloxone, oxycodone, methadone, ketanserin,tolmetin, ketoprofen, nabumetone, canrenone, canrenonate, mebendazole,oxolinic acid, tetracycline, chlortetracycline, oxytetracycline,demeclocycline, doxycycline, minocycline, daunorubicin, doxorubicin,mitoxantrone, plicamycin, mitomycin, indan-1,3-dione, anisindione,testosterone (and related C-17 esters, e.g., propionate, enanthate,cypionate), dihydrotesterone, cyproterone acetate, estrone,progesterone, medroxyprogesterone acetate, hydroxyprogesterone caproate,norethindrone, norethynodrel, megestrol acetate, norgestrel,mifepristone, methandrostenolone, oxandrolone, testolactone, cyproteroneacetate, prednisone, prednisolone, betamethasone, dexamethasone, other3-, 17-, or 20-ketosteroids (e.g., dehydroepiandorsterone,androstenedione, cortisol, cortisone, aldosterone, etc.), andparticularly β-lapachone compounds.

Another aspect of the present invention relates to methods for treatingcancer or other cell proliferative disorder comprising administering acomposition comprising a therapeutically effective amount of at leastone polymer-modified therapeutic agent, such as a β-lapachone compound,to a subject in need thereof. Another aspect of the invention relates tomethods for enhancing the accumulation of chemotherapeutic agents, suchas β-lapachone or an analog thereof, in a tumor tissue. Such a methodgenerally comprises providing a polymer-modified therapeutic agent, suchas β-lapachone or an analog thereof, and administering it to a patientor subject bearing a tumor tissue, thereby contacting the tumor tissuewith the agent where it may accumulate. In a preferred embodiment themethod is a method of enhancing the accumulation of chemotherapeuticagents in a tumor of patient. In preferred embodiments, the methods ofthe invention are particularly useful as methods for the treatment ofmammalian cancers, including, but not limited to, lung cancer, breastcancer, colon cancer, ovarian cancer, prostate cancer, multiple myelomaor malignant melanoma, or accumulation of therapeutic agents in suchcancer tissues. In other embodiments, the methods of treatment includetreatment of carcinoma, adenocarcinoma, and sarcoma. In more preferredembodiments the methods of treatment are methods of treating a human.Due to the ability to conjugate substantial amounts ofquinone-containing or carbonyl-containing therapeutic agents to thepolymeric vehicles employing the linking chemistries of the presentinvention the methods of the invention avoid the necessity of deliveringlarge amounts of solublizing agents, such as cyclodextrins, that areoften required to deliver therapeutic quantities of water-insolublecompounds, and which have their own toxicological issues.

According to the methods of the invention, the polymer-modifiedtherapeutic agents of the invention may be administered to a subject viaany suitable delivery route known in the art. Specific exemplaryadministration routes include peripheral and central routes such asintralymphatic (e.g., intra-lymph node), intravenous (bolus andinfusion), and intracerebral. Other routes such as oral, ocular rectal,buccal, topical, nasal, subcutaneous and intramuscular may be employed.In preferred embodiments the route of administration is parenteraladministration. In the most preferred embodiments the route ofadministration is intravenous.

As used herein the term “therapeutically effective amount” refers to anamount of a pharmaceutical agent to treat, ameliorate, or prevent theidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, ED₅₀/LD₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies may be used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include an ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

More specifically, the concentration-biological effect relationshipsobserved with regard to the therapeutics employed to form thepolymer-modified therapeutic agents of the present invention indicate aninitial target plasma concentration of the unconjugated therapeuticsranging from approximately 0.1 μg/ml to approximately 100 μg/ml,preferably from approximately 0.1 μg/ml to approximately 50 μg/ml, morepreferably the unconjugated therapeutics will have an initial targetplasma concentration from approximately 1 μg/ml to approximately 25μg/ml. To achieve such plasma concentrations, the polymer-modifiedtherapeutic agents of the present invention may be administered at dosesthat vary from 0.1 mg to 2,000 mg, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and is generally available topractitioners in the art. In general the dose of the therapeutic agentwill be in the range of about 0.1 mg/day to about 2 g/day, or about 0.5mg to about 2 g/day, or about 1 mg to about 1 g/day not including theweight of polymeric vehicle. The dose may be administered in a single,divided, or continuous doses for a patient weighing between about 40 toabout 100 kg (which dose may be adjusted for patients above or belowthis weight range, particularly children under 40 kg).

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage, androute of administration are adjusted to provide sufficient levels of theactive agent(s) or to maintain the desired effect. Factors that may betaken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

As used herein, a “subject” can be any mammal, e.g., a human, a primate,mouse, rat, dog, cat, cow, horse, pig, sheep, goat, camel. In apreferred aspect, the subject is a human.

As used herein, a “subject in need thereof” is a subject having a cellproliferative disorder, or a subject having an increased risk ofdeveloping a cell proliferative disorder relative to the population atlarge. In one aspect, a subject in need thereof has a precancerouscondition. In a preferred aspect, a subject in need thereof has cancer.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. In one aspect, a cell proliferativedisorder includes a non-cancerous condition, e.g., rheumatoid arthritis;inflammation; autoimmune disease; lymphoproliferative conditions;acromegaly; rheumatoid spondylitis; osteoarthritis; gout, otherarthritic conditions; sepsis; septic shock; endotoxic shock;gram-negative sepsis; toxic shock syndrome; asthma; adult respiratorydistress syndrome; chronic obstructive pulmonary disease; chronicpulmonary inflammation; inflammatory bowel disease; Crohn's disease;psoriasis; eczema; ulcerative colitis; pancreatic fibrosis; hepaticfibrosis; acute and chronic renal disease; irritable bowel syndrome;pyresis; restenosis; cerebral malaria; stroke and ischemic injury;neural trauma; Alzheimer's disease; Huntington's disease; Parkinson'sdisease; acute and chronic pain; allergic rhinitis; allergicconjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus. Inanother aspect, a cell proliferative disorder includes a precancer or aprecancerous condition. In another aspect, a cell proliferative disorderincludes cancer. Various cancers to be treated include but are notlimited to breast cancer, lung cancer, colorectal cancer, pancreaticcancer, ovarian cancer, prostate cancer, renal carcinoma, hepatoma,brain cancer, melanoma, multiple myeloma, chronic myelogenous leukemia,hematologic tumor, and lymphoid tumor, including metastatic lesions inother tissues or organs distant from the primary tumor site. Cancers tobe treated include but are not limited to sarcoma, leiomyosarcoma,carcinoma, and adenocarcinoma. In one aspect, a “precancer cell” or“precancerous cell” is a cell manifesting a cell proliferative disorderthat is a precancer or a precancerous condition. In another aspect, a“cancer cell” or “cancerous cell” is a cell manifesting a cellproliferative disorder that is a cancer. Any reproducible means ofmeasurement may be used to identify cancer cells or precancerous cells.In a preferred aspect, cancer cells or precancerous cells are identifiedby histological typing or grading of a tissue sample (e.g., a biopsysample). In another aspect, cancer cells or precancerous cells areidentified through the use of appropriate molecular markers.

In one aspect, a cancer that is to be treated has been staged accordingto the American Joint Committee on Cancer (AJCC) TNM classificationsystem, where the tumor (T) has been assigned a stage of TX, T1, T1mic,T1a, T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; and where the regionallymph nodes (N) have been assigned a stage of NX, N0, N1, N2, N2a, N2b,N3, N3a, N3b, or N3c; and where distant metastasis (M) has been assigneda stage of MX, M0, or M1. In another aspect, a cancer that is to betreated has been staged according to an American Joint Committee onCancer (AJCC) classification as Stage I, Stage IIA, Stage IIB, StageIIIA, Stage IIIB, Stage IIIC, or Stage IV. In another aspect, a cancerthat is to be treated has been assigned a grade according to an AJCCclassification as Grade GX (e.g., grade cannot be assessed), Grade 1,Grade 2, Grade 3 or Grade 4. In another aspect, a cancer that is to betreated has been staged according to an AJCC pathologic classification(pN) of pNX, pN0, PN0 (1−), PN0 (1+), PN0 (mol−), PN0 (mol+), PN1,PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

In one aspect, a cancer that is to be treated includes a tumor that hasbeen determined to be less than or equal to about 2 centimeters indiameter. In another aspect, a cancer that is to be treated includes atumor that has been determined to be from about 2 to about 5 centimetersin diameter. In another aspect, a cancer that is to be treated includesa tumor that has been determined to be greater than or equal to about 3centimeters in diameter. In another aspect, a cancer that is to betreated includes a tumor that has been determined to be greater than 5centimeters in diameter. In another aspect, a cancer that is to betreated is classified by microscopic appearance as well differentiated,moderately differentiated, poorly differentiated, or undifferentiated.In another aspect, a cancer that is to be treated is classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Inanother aspect, a cancer that is to be treated is classified bymicroscopic appearance as being associated with areas of necrosis (e.g.,areas of dying or degenerating cells). In one aspect, a cancer that isto be treated is classified as having an abnormal karyotype, having anabnormal number of chromosomes, or having one or more chromosomes thatare abnormal in appearance. In one aspect, a cancer that is to betreated is classified as being aneuploid, triploid, tetraploid, or ashaving an altered ploidy. In one aspect, a cancer that is to be treatedis classified as having a chromosomal translocation, or a deletion orduplication of an entire chromosome, or a region of deletion,duplication or amplification of a portion of a chromosome.

In one aspect, a cancer that is to be treated is evaluated by DNAcytometry, flow cytometry, or image cytometry. In one aspect, a cancerthat is to be treated has been typed as having 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% of cells in the synthesis stage of cell division(e.g., in S phase of cell division). In one aspect, a cancer that is tobe treated has been typed as having a low S-phase fraction or a highS-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder.” in one aspect, a normal celllacks unregulated or abnormal growth, or both, that can lead to thedevelopment of an unwanted condition or disease. Preferably, a normalcell possesses normally functioning cell cycle checkpoint controlmechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter, such as a polymer-modifiedtherapeutic agent, is in direct contact with a cell, or is close enoughto induce a desired biological effect in a cell.

As used herein, “candidate compound” refers to a compound of the presentinvention that has been or will be tested in one or more in vitro or invivo biological assays, in order to determine if that compound is likelyto elicit a desired biological or medical response in a cell, tissue,system, animal or human that is being sought by a researcher orclinician. In one aspect, in vitro or in vivo biological assays include,but are not limited to, enzymatic activity assays, electrophoreticmobility shift assays, reporter gene assays, in vitro cell viabilityassays, and the assays set forth in Examples 12-15.

As used herein, “monotherapy” refers to administration of a singleactive or therapeutic compound, such as a polymer conjugate containing asingle type of therapeutic agent, to a subject in need thereof.Preferably, monotherapy will involve administration of a therapeuticallyeffective amount of an active compound. Monotherapy may be contrastedwith combination therapy, in which a combination of multiple activecompounds is administered, preferably with each component of thecombination present in a therapeutically effective amount. In oneaspect, montherapy with a compound of the present invention is moreeffective than combination therapy in inducing a desired biologicaleffect.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition ordisorder.

In one aspect, treating a cancer of the present invention results in areduction in size of a tumor. A reduction in size of a tumor may also bereferred to as “tumor regression.” Preferably, after treatment, tumorsize is reduced by 5% or greater relative to its size prior totreatment; more preferably, tumor size is reduced by 10% or greater;more preferably, reduced by 20% or greater; more preferably, reduced by30% or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75% or greater. Size of a tumor may be measured by anyreproducible means of measurement. In a preferred aspect, size of atumor may be measured as a diameter of the tumor.

In another aspect, treating a cancer of the present invention results ina reduction in tumor volume. Preferably, after treatment, tumor volumeis reduced by 5% or greater relative to its size prior to treatment;more preferably, tumor volume is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75% or greater. Tumor volume may be measured by anyreproducible means of measurement.

In another aspect, treating a cancer of the present invention results ina decrease in number of tumors. Preferably, after treatment, tumornumber is reduced by 5% or greater relative to number prior totreatment; more preferably, tumor number is reduced by 10% or greater;more preferably, reduced by 20% or greater; more preferably, reduced by30% or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. Number of tumors may be measured by any reproduciblemeans of measurement. In a preferred aspect, number of tumors may bemeasured by counting tumors visible to the naked eye or at a specifiedmagnification. In a preferred aspect, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

In another aspect, treating a cancer of the present invention results ina decrease in number of metastatic lesions in other tissues or organsdistant from the primary tumor site. Preferably, after treatment, thenumber of metastatic lesions is reduced by 5% or greater relative tonumber prior to treatment; more preferably, the number of metastaticlesions is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75%. The number ofmetastatic lesions may be measured by any reproducible means ofmeasurement. In a preferred aspect, the number of metastatic lesions maybe measured by counting metastatic lesions visible to the naked eye orat a specified magnification. In a preferred aspect, the specifiedmagnification is 2×, 3×, 4×, 5×, 10×, or 50×.

In another aspect, treating a cancer of the present invention results inan increase in average survival time of a population of treated subjectsin comparison to a population receiving carrier alone. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.In a preferred aspect, an increase in average survival time of apopulation may be measured, for example, by calculating for a populationthe average length of survival following initiation of treatment with anactive compound, such as a polymer-modified therapeutic agent of theinvention. In another preferred aspect, an increase in average survivaltime of a population may also be measured, for example, by calculatingfor a population the average length of survival following completion ofa first round of treatment with an active compound such as apolymer-modified therapeutic agent of the invention.

In another aspect, treating a cancer of the present invention results inan increase in average survival time of a population of treated subjectsin comparison to a population of untreated subjects. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.In a preferred aspect, an increase in average survival time of apopulation may be measured, for example, by calculating for a populationthe average length of survival following initiation of treatment with anactive compound, such as a polymer-modified therapeutic agent of theinvention. In another preferred aspect, an increase in average survivaltime of a population may also be measured, for example, by calculatingfor a population the average length of survival following completion ofa first round of treatment with an active compound such as apolymer-modified therapeutic agent of the invention.

In another aspect, treating a cancer of the present invention results inincrease in average survival time of a population of treated subjects incomparison to a population receiving monotherapy with a drug that is nota compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.In a preferred aspect, an increase in average survival time of apopulation may be measured, for example, by calculating for a populationthe average length of survival following initiation of treatment with anactive compound, such as a polymer-modified therapeutic agent of theinvention. In another preferred aspect, an increase in average survivaltime of a population may also be measured, for example, by calculatingfor a population the average length of survival following completion ofa first round of treatment with an active compound, such as apolymer-modified therapeutic agent of the invention.

In another aspect, treating a cancer of the present invention results ina decrease in the mortality rate of a population of treated subjects incomparison to a population receiving carrier alone. In another aspect,treating cancer results in a decrease in the mortality rate of apopulation of treated subjects in comparison to an untreated population.In a further aspect, treating cancer results a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Ina preferred aspect, a decrease in the mortality rate of a population oftreated subjects may be measured by any reproducible means. In anotherpreferred aspect, a decrease in the mortality rate of a population maybe measured, for example, by calculating for a population the averagenumber of disease-related deaths per unit time following initiation oftreatment with an active compound, such as a polymer-modifiedtherapeutic agent of the invention. In another preferred aspect, adecrease in the mortality rate of a population may also be measured, forexample, by calculating for a population the average number ofdisease-related deaths per unit time following completion of a firstround of treatment with an active compound, such as a polymer-modifiedtherapeutic agent of the invention.

In another aspect, treating a cancer of the present invention results ina decrease in tumor growth rate. Preferably, after treatment, tumorgrowth rate is reduced by at least 5% relative to number prior totreatment; more preferably, tumor growth rate is reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. Tumor growthrate may be measured by any reproducible means of measurement. In apreferred aspect, tumor growth rate is measured according to a change intumor diameter per unit time.

In another aspect, treating a cancer of the present invention results ina decrease in tumor regrowth. Preferably, after treatment, tumorregrowth is less than 5%; more preferably, tumor regrowth is less than10%; more preferably, less than 20%; more preferably, less than 30%;more preferably, less than 40%; more preferably, less than 50%; evenmore preferably, less than 50%; and most preferably, less than 75%.Tumor regrowth may be measured by any reproducible means of measurement.In a preferred aspect, tumor regrowth is measured, for example, bymeasuring an increase in the diameter of a tumor after a prior tumorshrinkage that followed treatment. In another preferred aspect, adecrease in tumor regrowth is indicated by failure of tumors to reoccurafter treatment has stopped.

In another aspect, treating or preventing a cell proliferative disorderof the present invention results in a reduction in the rate of cellularproliferation. Preferably, after treatment, the rate of cellularproliferation is reduced by at least 5%; more preferably, by at least10%; more preferably, by at least 20%; more preferably, by at least 30%;more preferably, by at least 40%; more preferably, by at least 50%; evenmore preferably, by at least 50%; and most preferably, by at least 75%.The rate of cellular proliferation may be measured by any reproduciblemeans of measurement. In a preferred aspect, the rate of cellularproliferation is measured, for example, by measuring the number ofdividing cells in a tissue sample per unit time.

In another aspect, treating or preventing a cell proliferative disorderof the present invention results in a reduction in the proportion ofproliferating cells. Preferably, after treatment, the proportion ofproliferating cells is reduced by at least 5%; more preferably, by atleast 10%; more preferably, by at least 20%; more preferably, by atleast 30%; more preferably, by at least 40%; more preferably, by atleast 50%; even more preferably, by at least 50%; and most preferably,by at least 75%. The proportion of proliferating cells may be measuredby any reproducible means of measurement. In a preferred aspect, theproportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. In another preferred aspect, theproportion of proliferating cells is equivalent to the mitotic index.

In another aspect, treating or preventing a cell proliferative disorderof the present invention results in a decrease in size of an area orzone of cellular proliferation. Preferably, after treatment, size of anarea or zone of cellular proliferation is reduced by at least 5%relative to its size prior to treatment; more preferably, reduced by atleast 10%; more preferably, reduced by at least 20%; more preferably,reduced by at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. Size of an areaor zone of cellular proliferation may be measured by any reproduciblemeans of measurement. In a preferred aspect, size of an area or zone ofcellular proliferation may be measured as a diameter or width of an areaor zone of cellular proliferation.

In another aspect, treating or preventing a cell proliferative disorderof the present invention results in a decrease in the number orproportion of cells having an abnormal appearance or morphology.Preferably, after treatment, the number of cells having an abnormalmorphology is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. An abnormal cellular appearance ormorphology may be measured by any reproducible means of measurement. Inone aspect, an abnormal cellular morphology is measured by microscopy,e.g., using an inverted tissue culture microscope. In one aspect, anabnormal cellular morphology takes the form of nuclear pleiomorphism.

In one aspect, preventing cancer metastases results in a decrease innumber of tumors. Preferably, after treatment, tumor number is reducedby 5% or greater relative to number prior to treatment; more preferably,tumor number is reduced by 10% or greater; more preferably, reduced by20% or greater; more preferably, reduced by 30% or greater; morepreferably, reduced by 40% or greater; even more preferably, reduced by50% or greater; and most preferably, reduced by greater than 75%. Numberof tumors may be measured by any reproducible means of measurement. In apreferred aspect, number of tumors may be measured by counting tumorsvisible to the naked eye or at a specified magnification. In a preferredaspect, the specified magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

In another aspect, preventing cancer metastases results in a decrease innumber of metastatic lesions in other tissues or organs distant from theprimary tumor site. Preferably, after treatment, the number ofmetastatic lesions is reduced by 5% or greater relative to number priorto treatment; more preferably, the number of metastatic lesions isreduced by 10% or greater; more preferably, reduced by 20% or greater;more preferably, reduced by 30% or greater; more preferably, reduced by40% or greater; even more preferably, reduced by 50% or greater; andmost preferably, reduced by greater than 75%. The number of metastaticlesions may be measured by any reproducible means of measurement. In apreferred aspect, the number of metastatic lesions may be measured bycounting metastatic lesions visible to the naked eye or at a specifiedmagnification. In a preferred aspect, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. In oneaspect, the compared populations are cell populations. In a preferredaspect, a compound of the present invention, such as a polymer-modifiedtherapeutic agent of the invention or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof, actsselectively on a cancer or precancerous cell but not on a normal cell.In another preferred aspect, a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, acts selectively to modulate one molecular target(e.g., E2F-1) but does not significantly modulate another moleculartarget (e.g., Protein Kinase C). In another preferred aspect, theinvention provides a method for selectively inhibiting the activity ofan enzyme, such as a kinase. Preferably, an event occurs selectively inpopulation A relative to population B if it occurs greater than twotimes more frequently in population A as compared to population B. Morepreferably, an event occurs selectively if it occurs greater than fivetimes more frequently in population A. More preferably, an event occursselectively if it occurs greater than ten times more frequently inpopulation A; more preferably, greater than fifty times; even morepreferably, greater than 100 times; and most preferably, greater than1000 times more frequently in population A as compared to population B.For example, cell death would be said to occur selectively in cancercells if it occurred greater than twice as frequently in cancer cells ascompared to normal cells.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3d ed.), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18th edition (1990). Thesetexts can, of course, also be referred to in making or using an aspectof the invention.

The polymer-modified therapeutic agents of the inventions may becombined with one or more additional therapeutic agents when used as atherapeutic treatment. In one embodiment, where the polymer-modifiedtherapeutic has anti-tumor or anti-cancer activity, such as in the caseof β-lapachone compounds, the polymer-modified therapeutic agent may beadvantageously combined with one or more additional chemotherapeuticagents (second anti-cancer agents) useful in the treatment of cancer.The second chemotherapeutic agent can be a taxane, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, a targeted monoclonal or polyconal antibody,an inhibitor of a molecular target or enzyme (e.g., a kinase inhibitor),or a cytidine analogue drug. In preferred aspects, the chemotherapeuticagent can be, but not restricted to, tamoxifen, raloxifene, anastrozole,exemestane, letrozole, HERCEPTIN® (trastuzumab), GLEEVEC® (imatanib),TAXOL® (paclitaxel), cyclophosphamide, lovastatin, minosine, araC,5-fluorouracil (5-FU), methotrexate (MTX), TAXOTERE® (docetaxel),ZOLADEX® (goserelin), vincristin, vinblastin, nocodazole, teniposide,etoposide, GEMZAR® (gemcitabine), epothilone, navelbine, camptothecin,daunonibicin, dactinomycin, mitoxantrone, amsacrine, doxorubicin(adriamycin), epirubicin or idarubicin or agents listed inwww.cancer.org/docroot/cdg/cdg_(—)0.asp. In another aspect, the secondchemotherapeutic agent can be a cytokine such as G-CSF (granulocytecolony stimulating factor). In some embodiments of the invention, thetherapeutic agents and methods of treatment encompassed by thisinvention may comprise administration of a quinone-containing orcarbonyl-containing therapeutic agent in both polymer conjugated andunconjugated forms. In another embodiment, a polymer-modifiedtherapeutic agent of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, analog or derivative thereof, maybe administered in combination with standard chemotherapy combinationssuch as, but not restricted to, CMF (cyclophosphamide, methotrexate and5-fluorouracil), CAF (cyclophosphamide, adriamycin and 5-fluorouracil),AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin,and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, andpaclitaxel), or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil andprednisone). In yet another embodiment, a polymer-modified therapeuticagent of the present invention, or a pharmaceutically acceptable salt,prodrug, metabolite, analog or derivative thereof, may be administeredin combination with radiation therapy. The skilled artisan willrecognize that a variety of other therapeutic agents in addition tothose recited above may be administered in combination with thepolymer-modified therapeutic agents of the present invention, and thatthey may act to augment or synergistically enhance the activity ofpolymer-modified therapeutic agents such as the polymer-modifiedβ-lapachone compounds of the invention.

The polymer-modified therapeutic agents of the present invention whenadministered in combination with another therapeutic agent may bedelivered in a unitary dosage form, or in separate dosage forms intendedfor simultaneous or sequential administration to a patient in need oftreatment. When administered sequentially, the combination may beadministered in two or more administrations. In an alternativeembodiment, it is possible to administer one or more polymer-modifiedtherapeutic agents of the present invention and one or more additionalactive ingredients by different routes. According to the methods of theinvention, the combination of active ingredients may be (1)co-formulated and administered or delivered simultaneously in a combinedformulation, (2) delivered by alternation or in parallel as separateformulations, or (3) by any other combination therapy regimen known inthe art. When delivered in alternation therapy, the methods of theinvention may comprise administering or delivering the activeingredients sequentially, e.g., in separate solution, emulsion,suspension, tablets, pills or capsules, or by different injections inseparate syringes. In general, during alternation therapy, an effectivedosage of each active ingredient is administered sequentially, i.e.,serially, whereas in simultaneous therapy, effective dosages of two ormore active ingredients are administered together. Various sequences ofintermittent combination therapy may also be used.

G. Pharmaceutical Compositions of the Invention

While it is possible for the polymer-modified therapeutic agents of thepresent invention, including the polymer-modified β-lapachone compoundsof the present invention, to be administered without admixture ordilution, it may be preferable to formulate the compounds aspharmaceutical compositions. As such, pharmaceutical compositionscomprising the polymer-modified therapeutic agents of the invention areprovided. These pharmaceutical compositions are useful in the methods ofthe invention, including but not limited to treatment of various cancersand enhancing the accumulation of chemotherapeutic agents, such asβ-lapachone or an analog thereof, in a tumor tissue. The pharmaceuticalcompositions of the invention may be formulated with pharmaceuticallyacceptable excipients such as carriers, solvents (including water orsaline), stabilizers, adjuvants, diluents, etc., depending upon theparticular mode of administration and dosage form. The pharmaceuticalcompositions should generally be formulated to achieve a physiologicallycompatible pH, and may range from a pH of about 5 to a pH of about 9, ormore preferably from about pH 6 to about pH 8 depending on theformulation and route of administration. In alternative embodiments, itmay be preferred that the pH is adjusted to a range from about pH 5.5 toabout pH 7.5 or from about 6.5 to 8.5.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compounds ofthe present invention. The term refers to any pharmaceutical excipientthat may be administered without undue toxicity. Pharmaceuticallyacceptable excipients are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the composition. Accordingly, there exists a wide varietyof suitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences). Suitableexcipients include, but are not limited to, carrier molecules thatinclude large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, polyethylene glycols (e.g., PEG 400) andinactive virus particles. Other exemplary excipients includeantioxidants such as ascorbic acid; chelating agents such as EDTA,carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose, liquids such as oils, water, saline,glycerol wetting or emulsifying agents; pH buffering substances; and thelike. Liposomes are also included within the definition ofpharmaceutically acceptable excipients.

The pharmaceutical compositions of the invention comprise atherapeutically effective amount of at least one polymer conjugate,together with one or more pharmaceutically acceptable excipients. In apreferred embodiment, comprising polymer conjugates capable ofdelivering β-lapachone, or a derivative or analog thereof, or apharmaceutically acceptable salt thereof, or a metabolite thereof, thecompositions preferably maintain a plasma concentration of unconjugatedβ-lapachone from approximately 0.1 μg/ml to approximately 100 μg/ml,preferably from approximately 0.1 μg/ml to approximately 50 μg/ml, morepreferably the unconjugated therapeutics will have an initial targetplasma concentration from approximately 1 μg/ml to approximately 25μg/ml. In another aspect, the pharmaceutical composition can maintain asuitable plasma concentration of β-lapachone for at least a month, atleast a week, at least 24 hours, at least 12 hrs, at least 6 hrs, atleast 1 hour. In a further aspect, a suitable plasma concentration ofβ-lapachone can be maintained indefinitely by the administration ofperiodic or continuous does of a suitable polymer conjugate. In yetanother aspect, the subject can be exposed to the pharmaceuticalcomposition in a AUC (area under the curve) range of about 0.5 μM-hr toabout 100 μM-hr, about 0.5 μM-hr to about 50 μM-hr, about 1 μM-hr toabout 25 μM-hr, about 1 μM-hr to about 10 μM-hr; about 1.25 μM-hr toabout 6.75 μM-hr, about 1.5 μM-hr to about 6.5 μM-hr, where theconcentration represents the total concentration of β-lapachone, or aderivative or analog thereof, or a pharmaceutically acceptable saltthereof, or a metabolite thereof. The pharmaceutical composition ofsuitable polymer conjugate, or a derivative or analog thereof, or apharmaceutically acceptable salt thereof, or a metabolite thereof can beadministered at a dosage from about 2 mg/m² to 5000 mg/m² per day, morepreferably from about 20 mg/m² to 2000 mg/m² per day, more preferablyfrom about 20 mg/m² to 500 mg/m² per day, most preferably from about 30to 300 mg/m² per day. Preferably, 2 mg/m² to 5000 mg/m² per day is theadministered dosage for a human. In another aspect, the pharmaceuticalcomposition can be administered at a dosage from about 0.001 to 100 mgof β-lapachone, or a derivative or analog thereof, or a pharmaceuticallyacceptable salt thereof, or a metabolite thereof per kilogram bodyweight of recipient per day; preferably about 0.01 to 50 mg per kilogrambody weight of recipient per day, more preferably from about 0.1 to 25mg per kilogram body weight of recipient per day, most preferably fromabout 1 to 15 mg per kilogram body weight of recipient per day. One ofordinary skill in the art can determine the appropriate dosage amount inmg/m² per day or mg per kilogram body weight of recipient per daydepending on subject to which the pharmaceutical composition is to beadministered. Pharmaceutical compositions of the invention capable ofdelivering β-lapachone, or a derivative or analog thereof, or apharmaceutically acceptable salt thereof, or a metabolite thereof at theabove-indicated ranges may be prepared from polymer conjugatedtherapeutics. Where the pharmaceutical compositions of the inventioncomprise a combination of polymer-modified therapeutic, such as aβ-lapachone compound, and a second therapeutic agent, such as achemotherapeutic useful in the treatment of cancer, the therapeuticamounts of the second agents are may be generally known in the art ormay be determined by the skilled clinician.

Some properties of polymer-modified β-lapachone compositions areprovided in Table 1.

TABLE 1 PGA Loading Free (kD) Loading by Wt. β-lapachone (Sodium by Wt.(%) UV by Wt. (%) Salt Conjugate Sample Salt) (%) (305 nm) (HPLC) Form

A 17.1 16.8 ± 0.6 N/A N/A Sodium salt

B C D E F G H I 17.1 17.1 56   56   56   56   56   56   19.3 ± 0.3 25.5± 1.9 18.6 ± 2.0 32.8 ± 1.2 31.9 ± 1.6 33.1 ± 0.2 36.1 ± 1.9 37.1 ± 0.8N/A 28.8 ± 0.34 25.0 ± 0.4  34.5 ± 0.5  30.0 ± 0.7  33.6 ± 0.4  36.6 ±1.4  38.5 ± 1.2  0.4-0.58 0.1 0.4 0.3 0.1 0.4 0.5 0.4 Free acid Freeacid Free acid Free acid Free acid Free acid Free acid Free acid

J K  7.1 56   16.2 ± 0.1 15.3 ± 0.7 N/A N/A N/A N/A Sodium salt Sodiumsalt N/A is not available

The pharmaceutical compositions of the invention may be formulated inany form suitable for the intended method of administration. Preferredformulations of the present invention are those for parenteraladministration, and in particular intravenous administration. Suchpharmaceutical compositions include liquid solutions, emulsions, orsuspensions. In a more preferred embodiment, pharmaceutical compositionsof the invention may also be formulated as a lyophilized solid, whichprovides a convenient stable form that is reconstituted with aphysiologically compatible solvent, such as water or saline, prior toadministration either parenterally or by any other suitable route.

In other embodiments, pharmaceutical compositions of the invention maybe formulated as suspensions comprising a compound of the presentinvention in admixture with at least one pharmaceutically acceptableexcipient suitable for the manufacture of a suspension. In yet anotherembodiment, pharmaceutical compositions of the invention may beformulated as dispersible powders and granules suitable for preparationof a suspension by the addition of suitable excipients.

Where routes of administration such as oral, ocular rectal, buccal,topical, nasal, subcutaneous or and intramuscular are deemed to bedesirable, pharmaceutical compositions of the invention may beformulated as syrups, creams, ointments, tablets, and the like employingsuitable pharmaceutical excipients known in the art.

All publications mentioned in the above specification are hereinincorporated by reference. The above description and drawings areillustrative of preferred embodiments of the present invention. It isnot intended that the present invention be limited to the illustrativeembodiments. Any modification of the present invention within the spiritand scope of this disclosure should be considered a part of the presentinvention.

To assist in understanding the present invention, the following Examplesare included.

EXAMPLES

The present invention is described in more detail with reference to thefollowing non-limiting examples, which are offered to more fullyillustrate the invention, but are not to be construed as limiting thescope thereof. The examples illustrate the preparation of certaincompounds of the invention, and the testing of these compounds in vitroand/or in vivo. Those of skill in the art will understand that thetechniques described in these examples represent techniques described bythe inventors to function well in the practice of the invention, and assuch constitute preferred modes for the practice thereof. However, itshould be appreciated that those of skill in the art should in light ofthe present disclosure, appreciate that many changes can be made in thespecific methods that are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Preparation of2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl 4-nitrobenzoate

To a solution of 4-nitrobenzoylchloride (4.0 g, 21.6 mmoles) andN-(9-fluorenyl-methoxycarbonyl)ethanolamine (7.3 g, 25.9 mmoles) in 160ml of methylene chloride is added triethylamine (3.1 ml, 22.6 mmoles).The mixture is stirred at room temperature for one hour, then is dilutedwith 400 ml of methylene chloride. The organic solution is washed withtwo portions of 200 ml of water and one portion of 200 ml of a saturatedaqueous solution of sodium chloride. The organic phase is dried withNa₂SO₄, filtered and concentrated in vacuo. The resulting pale yellowsolid is triturated in ethyl acetate. Filtration afforded 7.347 g of thetitle compound (79%). 400 MHz ¹H NMR (DMSO-d₆) δ: 8.32 (d, J=8.4 Hz,2H), 8.20 (d, J=8.8 Hz, 2H), 7.87 (d, J=7.3 Hz, 2H), 7.66 (d, J=7.3 Hz,2H), 7.61 (t, J=5.7 Hz, 1H), 7.39 (t, J=7.5 Hz, 2H), 7.28 (t, J=7.5 Hz,2H), 4.4-4.3 (m, 4H), 4.20 (t, J=6.8 Hz, 1H), 3.45-3.35 (m, 2H). LCMS:433 [M+H].

Example 2 Preparation of2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl 4-aminobenzoate

A solution of 2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl4-nitrobenzoate (7.47 g, 17 mmoles) in 1 liter of ethyl acetate,containing 1.5 g of 10% Pd on carbon (wet) is evacuated and filled withH₂ twice. The mixture is subjected to hydrogen at 1 atmosphere ofpressure for two hours. The catalyst is filtered off on a Celite pad andthe solution is concentrated in vacuo, yielding a yellow oil.Purification on silica gel using a gradient of 30-45% ethyl acetate inhexanes provided 5.436 g (80%) of the title compound as a white solid.400 MHz H NMR (CDCl₃) δ: 7.86 (d, J=8.4 Hz, 2H), 7.75 (d, J=7.3 Hz, 2H),7.58 (d, J=7.3 Hz, 2H), 7.39 (t, J=7.3 Hz, 2H), 7.30 (t, J=7.5 Hz, 1H),6.64 (d, J=8.4 Hz, 2H), 5.13 (br. t, 1H), 4.40 (d, J=7.0 Hz, 2H), 4.36(t, J=5.1 Hz, 2H), 4.22 (t, J=7.0 Hz, 1H), 4.1-4.0 (br. s, 2H),3.65-3.55 (m, 2H). LCMS: 403 [M+H].

Example 3 Preparation of2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl4-{[(6Z)-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]chromen-6(5H)-ylidene]amino}benzoate

A solution of 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione(2.975 g, 12.3 mmoles) in 120 ml of methylene chloride is treated with6.14 ml of a 1.0 M solution of TiCl₄ in methylene chloride. The mixtureis stirred at room temperature for 5 minutes, then a solution of2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl 4-aminobenzoate (5.436g, 13.5 mmoles) in 60 ml of methylene chloride is introduced followed bytriethylamine (10.3 ml, 73.7 mmoles). The mixture is stirred at roomtemperature for 10 minutes. A second portion of 6.14 ml of a 1.0 Msolution of TiCl₄ in methylene chloride is added and the mixture isstirred at room temperature for 10 minutes. A third portion of 6.14 mlof a 1.0 M solution of TiCl₄ in methylene chloride is added and themixture is stirred at room temperature for 15 minutes. After stirringwith the third aliquot of TiCl₄, the mixture is poured onto 300 ml ofice, to form a mixture having a precipitate. The mixture is filtered toremove the precipitate and the organic and aqueous phases are thenseparated. The aqueous phase is extracted with two portions of 200 ml ofmethylene chloride. The combined organic extracts are dried with Na₂SO₄,filtered and concentrated in vacuo, giving a brown oil. Purification onsilica gel using a gradient of 0-10% ethyl acetate in methylene chlorideprovided a brown reddish foam, which is triturated in diethyl ether,yielding 990 mg (13%) of the title compound as a brown solid. M.p.:163-164° C. 400 MHz ¹H NMR (DMSO-d₆) δ: 8.13 (d, J=7.3 Hz, 1H), 7.93 (d,J=8.4 Hz, 2H), 7.88 (d, J=7.7 Hz, 2H), 7.82 (d, J=8.1 Hz, 1H), 7.7-7.55(m, 5H), 7.40 (t, J=7.5 Hz, 2H), 7.30 (t, J=7.5 Hz, 2H), 6.79 (d, J=8.4Hz, 2H), 4.33 (d, J=7.0 Hz, 2H), 4.3-4.2 (m, 3H), 3.45-3.35 (m, 2H),2.25 (t, J=6.4 Hz, 2H), 1.79 (t, J=6.4 Hz, 2H), 1.41 (s, 6H). LCMS: 627[M+H]. Calc. for C₃₉H₃₄N₂O₆ 0.1 water: C, 74.46; H, 5.48; N, 4.46. FoundC, 74.48; H, 5.06; N, 4.43.

Example 4 Preparation ofN-{2-[(4-{[(6Z)-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]-chromen-6(5H)-ylidene]amino}benzoyl)oxy]ethyl}γ-poly-L-glutamate

A solution of 2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl4-{[(6Z)-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]chromen-6(5H)-ylidene]amino}benzoate(150 mg, 0.239 mmoles) in 7 ml of DMF is treated with1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (150 μl, 1 mmole). The mixtureis stirred at room temperature for two hours. The mixture is thendiluted with 5 ml of DMF and polyglutamic acid (17,000 Da) is added (309mg, 2.3 mmole) followed by triethylamine (333 μl, 2.3 mmoles) and HBTU(907 mg, 2.3 mmoles). The mixture is stirred at room temperatureovernight. The mixture is poured into 100 ml of a 0.5 M aqueous solutionof sodium bicarbonate, stirred for 30 minutes, then filtered. Thesolution is freeze-dried and the resulting solid is submitted todialysis using molecular membrane against 3 changes of water. Thedialyzed solution is freeze-dried to yield 78 mg of a dark brown solid.The quantity of 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dionebound to polyglutamic acid is determined by release of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione using 0.1 N HCland UV detection to be 16.9% by weight based upon the weight of polymerconjugate hydrolyzed.

Example 5a Synthesis of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylN-(tert-butoxycarbonyl)glycinate

A mixture of zinc dust (6.0 g, 91.7 mmol),2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (6.0 g, 24.8mmol), Na₂S₂O₄ (17.26 g, 99.1 mmol), N-(tert-butoxycarbonyl)glycine(8.77 g, 49.6 mmol), triethylamine (3.1 ml, 22.3 mmol), HBTU (18.79 g,49.6 mmol), and DMF (100 ml) is stirred for 16 hours at roomtemperature. To the reaction mixture is then added ethyl acetate (300ml). The reaction is filtered and the filtrate washed with H₂O (4×200ml). The organic extract is dried with Na₂SO₄, and concentrated underreduced pressure to yield a residue. The residue is dissolved in aceticanhydride (30 ml), zinc dust (3.0 g, 45.9 mmol) and triethylamine (3.35ml, 24.0 mmol) are then added. The mixture is heated to 90° C. withvigorous stirring and held at 90° C. for 2 hours, after which it isallowed to cool and the solvent is removed under reduced pressure toyield a second residue. The second residue is dissolved in ethyl acetate(200 ml) and washed with water (2×100 ml). The organic extract is driedwith Na₂SO₄ and concentrated under reduced pressure to yield unrefinedproduct. The unrefined product is purified by flash columnchromatography on silica, eluting with 2% ethyl acetate indichloromethane, to afford product about 60% pure. The product isfurther purified by crystallization from ethyl acetate/hexane, whichgives the desired product as pure white solid (3.2 g, 31%). M.p.=177°C.; 400 MHz ¹H NMR (CDCl₃) δ: 8.21 (d, J=8.4 Hz, 1H), 7.67 (d, J=8.4 Hz,1H), 7.46 (m, 2H), 5.13 (br. s, 1H), 4.30 (d, J=5.6 Hz, 2H), 2.68 (t,J=6.6 Hz, 2H), 2.38 (s, 3H), 1.87 (t, J=6.6 Hz, 2H), 1.48 (s, 9H), 1.42(s, 6H); LCMS: 444 [M+H]; Calc. for C₂₄H₂₉NO₇: C, 64.94; H, 6.59; N,3.16. Found C, 64.98; H, 6.51; N, 3.15.

Example 5b Synthesis of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylN-(tert-butoxycarbonyl)glycinate (1)

To a mixture of zinc dust (50.0 g, 0.765 mol) and2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (50.0 g, 0.206mol) is added DMF (500 mL). The mixture is allowed to stir for 15 min.To the reaction is then added a mixture of N-(tert-butoxycarbonyl)glycine (43.38 g, 0.247 mol) and HBTU (156.5 g, 0.413 mol) in 5 smallportions over a period of 3 hours to control the exothermic reaction.The reaction was stirred for 16 hours. Ethyl acetate (EtOAc) (1300 mL)is then added, and the reaction is filtered through celite. The celiteis bed washed with EtOAc (200 mL). The combined EtOAc solutions arewashed with water (2×1000 mL) followed by saturated NaCl (500 mL). Theorganic layer is dried over sodium sulfate and concentrated underreduced pressure. The crude residue (103 g) is dissolved in aceticanhydride (150 mL) followed by the addition of zinc dust (13.5 g, 0.206mol) and triethylamine (25.9 mL, 0.186 mol). The reaction is stirred for1.15 hours. The solvent (acetic anhydride and triethylamine) is removedunder reduced pressure to yield a residue. The residue was dissolved inEtOAc (1000 mL) and washed with water (2×500 mL), 5% sodium bicarbonate(2×500 mL) saturated NaCl (500 mL) and dried over Na₂SO₄. Concentrationof the ethyl acetate solution under reduced pressure yields a yellowishwhite crude product (103 g) that is purified by crystallization fromhexane-ethyl acetate. For crystallization, the crude product isdissolved in refluxing EtOAc (200 mL) and hexane (300 mL) is added. Themixture is allowed to cool to room temperature and stirred for 14 hr. Asolid separated out which is collected by filtration and washed with 5%EtOAc in hexane (500 mL). The desired product (38.2 g) is obtained 98%pure with 2% of bis-glycine as a side-product. The 98% pure product(38.2 g) is again crystallized from refluxing EtOAc (165 mL) and hexanes(250 mL). The hot solution is allowed to cool to room temperature andstirred for 15 hr. The white solid obtained is filtered, washed with 5%EtOAc in hexane (400 mL), and dried under high vacuum at 45-50° C. Thedesired product (34.6 g, 38%) is obtained as a white solid at 99.83%purity based upon HPLC analysis. The ¹H NMR is the same as described inExample 5a.

Example 6a Synthesis of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinatehydrochloride

To a solution of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylN-(tert-butoxycarbonyl)glycinate (1) (2.35 g, 4.0 mmol) in 1,4-dioxane(25 ml) is added a solution of hydrogen chloride gas in anhydrous1,4-dioxane (4.0 M, 60 ml). The reaction is stirred at room temperaturefor 6 hours. Upon drying under reduced pressure, the producthydrochloride salt is obtained as a white solid (1.489 g, 95%).M.p.=176-178° C.; 400 MHz ¹H NMR (DMSO-d₆) δ: 8.58 (br. s, 3H), 8.14 (m,1H), 7.9 (m, 1H), 7.55 (m, 2H), 4.41 (s, 2H), 2.62 (t, J=6.6 Hz, 2H),2.41 (s, 3H), 1.88 (t, J=6.6 Hz, 2H), 1.39 (s, 6H); LCMS: 344 [M+H];Calc. for C₁₉H₂₁NO₅ 1.25HCl: C, 58.62; H, 5.77; N, 3.6. Found C, 58.7;H, 5.72; N, 3.47.

Example 6b Synthesis of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinatehydrochloride

To a solution of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2,1-benzo[h]chromen-6-ylN-(tert-butoxycarbonyl)glycinate (34.0 g, 76.6 mmol) in isopropylacetate (150 mL) is added concentrated hydrochloric acid (28.0 ml, 37%by weight, 12.1 M). The reaction is stirred at room temperature for 18hours. The solid is collected by filtration, washed with isopropylacetate (4×100 mL) and dried under high vacuum. The product is obtainedas an off-white solid (21.0 g, 73%) M.p.=190-193° C.; 400 MHz ¹H NMR(DMSO-d₆) δ: 8.76 (br. s, 3H), 8.14-8.11 (1H, m), 7.95-7.92 (1H, m),7.60-7.50 (2H, m), 4.39 (2H, s), 2.63 (2H, t, J=6.4 Hz), 2.43 (3H, s),1.87 (2H, t, J=6.4 Hz), 1.39 (6H, s); LCMS: 344 [M+H]; Calc. forC₁₉H₂₁NO₅ 1.07 HCl: C, 59.68; H, 5.83; N, 3.69. Found C, 59.66; H, 5.83;N, 3.69.

Example 7a Synthesis of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate

To a mixture of poly-L-glutamic acid (average molecular weight 17,000 Daby viscosity, 8,853 Da by GPC mALLS) (0.75 g, 5.81 mmol),5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinatehydrochloride (2) (0.481 g, 1.28 mmol), 4-(dimethylamino) pyridine(DMAP), and HBTU (0.58 g, 1.55 mmol) in DMF (6.0 ml) is added triethylamine (0.206 g, 2.04 mmol). The reaction is allowed to stir for 16hours. Water (10 ml) and 1.0 N HCl (20 ml) is added to the reactionmixture, which results in a white solid separating out. The solid iscollected by centrifugation, and the supernatant discarded. The solid iswashed 5 times with 1.0 N HCl (40 ml) and then with 5 times withdeionized water (40 ml), or until the pH of solution is between 4 and4.5. The solid is then suspended in 20 ml of water and lyophilized todryness, yielding a white powder (0.885 g, 76%). The quantity of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione bound to thepolymer is calculated by releasing2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione from theconjugate by treatment with 0.01 N NaOH (18.6+/−0.6% by weight basedupon the weight of the polymer conjugate hydrolyzed). 400 MHz ¹H NMR(DMSO-d₆) δ: 12.1 (br. s, 1.81H), 8.6-8.4 (br. s, 1H), 8.4-7.9 (br s,3.5H), 7.9-7.65 (br s, 1H), 7.65-7.2 (br s, 2H), 4.4-3.8 (br m, 4.2H),2.8-2.6 (br s, 3H), 2.5-1.6 (br m, 15H), 1.3 (br s, 6H). The quantity of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione conjugated to thepolymeric vehicle may be determined as described in Example 11

Example 7b Synthesis of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate

To poly-L-glutamic acid (average molecular weight: 47.9 kD by viscosity,37.83 kD by GPC mALLS (multi-angle laser light scattering)) (1.0 g, 7.75mmol) is added DMF (100 mL) and the suspension allowed to stir for 30min. To the suspension is then added HBTU (0.7141 g, 1.88 mmol) and thereaction allowed to stir for 10 min. This is followed by thesimultaneous addition of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinatehydrochloride (2) (0.625 g, 1.64 mmol) and dimethylaminopyridine (0.20g, 1.64 mmol); triethylamine (0.206 g, 2.04 mmol) is then added and thereaction is stirred for 16 hours. The solvent, including DMF, is thenremoved under reduced pressure to yield an oily residue, to which 1.0 NHCl (100 ml) is added resulting in a light yellow colored filament likesolid separating out. The solid is collected by centrifugation and thesupernatant discarded. The solid is washed 5 times with 1.0 N HCl (40mL) and then washed 5 times with deionized water (40 mL) or until the pHof solution is between 4 and 4.5. The solid is frozen and lyophilized todryness to yield a light yellow powder (1.12 g, 73%). Loading (18.6+/−2%by wt.) is calculated by releasing2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione from theconjugate by treatment with 0.01 N NaOH. 400 MHz ¹H NMR (DMSO-d₆) δ:12.1 (br. s, 2.3H), 8.6-8.4 (br. s, 0.5H), 8.3-7.9 (br s, 3.6H),7.9-7.65 (br s, 1.4H), 7.65-7.2 (br s, 2.1H), 4.5-4.0 (br m, 5.2H),2.7-2.5 (br s, 2.0H), 2.5-2.1 (br m, 9.2H), 2.1-1.6 (br m, 8.31H), 1.37(br s, 6H). The quantity of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione conjugated to thepolymeric vehicle may be determined as described in Example 11

Example 8 Synthesis of5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylglycinyl-poly(styrene-co-maleamide)

To a solution of styrene maleic anhydride (mol. wt. 1600 by GPC, 0.20 g,0.86 mmol) in THF is added sodium bicarbonate (0.288 g, 3.42 mmol)followed by5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinatehydrochloride (0.423 g, 1.15 mmol). The reaction is allowed to stir for16 hour at 40° C. The solvent is removed under reduced pressure to yielda residue, which is dissolved in dichloromethane (5 mL) and filteredthrough a plug of glass wool. To the clear dichloromethane filtrate isadded diethyl ether (100 mL), which results in a light yellow solidseparated out. The light yellow solid was filtered, washed with diethylether (50 mL), and dried under high vacuum to give the desired product(0.29 g, 52%). Loading (34-41% by wt.) is calculated by releasing2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione from theconjugate by treating it with 0.01 N NaOH. 400 MHz ¹H NMR (DMSO-d₆) δ:8.2-8.0 (br s, 1.2H), 8.0-7.7 (br s, 1H), 7.7-7.35 (br s, 1.56H),7.3-6.4 (br s, 4.5H), 4.4-3.8 (br m, 1.76H), 2.8-2.0 (br m, 4.5H),2.0-1.6 (br m, 2.9H), 1.5-1.1 (br m, 6H).

Example 9a Preparation of4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one

To a solution of 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione(1.21 g, 5 mmol) in benzene (12 ml) is added glycerol (0.92 g, 10 mmol)and p-toluenesulfonic acid monohydrate (0.28 g, 1.5 mmol). The resultingmixture is refluxed for 6 hours. After removal of the solvent, productis purified by column chromatography on silica gel eluting with 30%ethyl acetate in methylene chloride to provide 0.57 g (36% yield) as alight yellow solid, which is a mixture of diasteromers. M.p. 44-46° C.;400 MHz ¹H NMR (CDCl₃) δ: 1.41 (s, 6H), 1.73-1.82 (m, 2H), 2.36-2.42 (m,1H), 2.48-2.54 (m, 1H), 3.72-3.78 (m, 1H), 4.18 (d, J=2.5 Hz, 1H),4.20-4.29 (m, 1H), 4.40 (t, J=8.0 Hz, 1H), 4.71-4.74 (m, 1H), 4.85 (d,J=8.8 Hz, 1H), 7.38-7.44 (m, 2H), 7.54-7.56 (m, 1H), 7.75-7.78 (m, 1H);LCMS: Calc. for C₁₈H₂₁O₅: 217; Found 217.

Example 9b Preparation of4′-(azidomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-oneand its conversion into4′-(aminomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-oneStep 1. Synthesis of4′-(azidomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one

To a solution of4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one(316 mg, 1.0 mmol) in dichloromethane (2 mL) is added methanesulfonylchloride (0.095 mL, 1.3 mmol) and triethylamine (0.163 mL, 1.24 mmol).The mixture is stirred at room temperature for 2 hours. The reactionmixture is washed with water (2×1.0 mL), dried over sodium sulfate andconcentrated to provide(2,2-dimethyl-5-oxo-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′-yl)methylmethanesulfonate as a white solid (99% by LC/MS, UV 254 nm). The(2,2-dimethyl-5-oxo-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′-yl)methylmethanesulfonate (197 mg, 0.5 mmol) is dissolved in DMF (2 mL) followedby the addition of sodium azide (97 mg, 1.5 mmol). The reaction isheated at 80° C. for 24 hour and 100° C. for 12 h. The mixture isdiluted with CH₂Cl₂ (4 mL), washed with water (2 ml) and concentrated.The organic layer is dried with sodium sulfate, concentrated underreduced pressure and purified by flash column chromatography (SiO₂, 30%EtOAc in hexane) to provide4′-(azidomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one(25 mg, 15%) as a white solid. 400 MHz ¹H NMR (CDCl₃) δ: 7.78-7.56 (m,1H), 1.60-7.57 (m, 1H), 7.44-7.40 (m, 2H), 4.68-4.62 (m, 1H), 4.46-4.42(m, 1H), 4.23 (t, J=8 Hz, 1H). 3.96-3.91 (m, 1H), 3.54-3.49 (m, 1H),2.48-2.42 (m, 2H), 1.79 (t, J=6.8 Hz, 2H), 1.42 (d, J=3.2 Hz, 6H); LCMS:342 [M+H].

Step 2. Conversion of4′-(azidomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-oneto4′-(aminomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one

To a solution of4′-(azidomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one(0.012 mg, 0.035 mmol) in THF (1.0 mL) is added triphenylphosphine(0.033 mg, 0.13 mmol). The mixture is stirred at room temperature for 3hour followed by addition of water (50 μL). The reaction is then stirredfor another 16 hour at room temperature and solvent is removed underreduce pressure to yield a reside of4′-(aminomethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-oneas confirmed by LC/MS, which indicates the product, which has amolecular weight of 315 Da, is present in the crude residue along withtriphenylphosphine oxide (a side product from the triphenylphosphine).

Example 10 Preparation of(2,2-dimethyl-5-oxo-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′-yl)methylγ-poly-L-glutamate sodium

To a solution of poly-L-glutamic acid (Mw approximately 17,000 Da) (170mg) in DMF (5 ml) is added the ketal linker conjugated2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione product preparedin Example 9a (63 mg, 0.20 mmol), 1,3-dicyclohexylcarbodiimide (144 mg,0.70 mmol), and 4-dimethylaminopyridine (5 mg, 0.04 mmol). The mixtureis stirred at room temperature overnight. The progress of the reactionmay be monitored by HPLC for disappearance of the ketal linkerconjugate. Typical HPLC conditions employ an Aligent Zorbax SB-C8 rapidresolution cartridge (30×4.6 mm, 3.5 μm) fitted with a Phenomenex C8(Octyl, MOS) guard column (4.00×3.00 mm cartridge) with UV detection at254 nm. The column is equilibrated at a flow rate of 3 ml/min in amixture of 95% solvent A (water with 0.1% TFA) and 5% solvent B(acetonitrile with 0.1% TFA), after sample application, the solvent ischanged to 5% solvent A and 95% solvent B at 0.25 minutes, after 4.1minutes the column is returned to equilibration buffer (95% solvent Aand 5% solvent B) for 4.8 minutes. When the ketal linker conjugate hasbeen consumed, the resulting mixture is diluted with chloroform (10 ml)and a solution of 0.5 M sodium bicarbonate (15 ml) is added. The mixtureis shaken well and subject to centrifugation. The chloroform layer isremoved and the water layer is washed three times with 5 ml ofchloroform in each wash. The resulting aqueous solution is dialyzedemploying a 6000-8000 molecular weight cut-off dialysis membrane againstdeionized water. Dialysis employed six changes deionized water replacedat 4 hours intervals over 24 h. Lyophilization of the aqueous solutionyielded 170 mg of product as a white soft solid. M.p. 265-268° C.

The quantity of ketal linked2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (β-lapachone)associated with polyglutamic acid is determined by hydrolysis of thecompound from the polymer followed by quantitation. To a solution ofconjugate (1.4 mg) in water (0.3 ml) is added 50% NaOH (100 μl). Themixture is sonicated at room temperature for 5 minutes.

Product is extracted with ethyl acetate (1.0 ml×3). Organic extracts arecombined and solvent is removed. The residue is dissolve in 1.0 ml DMSO.Released ketal linker conjugated therapeutic agent is determined by HPLCas described above. The quantity of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione bound to thepolymer is calculated to be 18.5% by weight based upon the weight of thepolymer conjugate hydrolyzed.

Alternatively, 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione(β-lapachone) is released from the PGA ketal conjugate in 1N HCl at 37°C.,

and the β-lapachone extracted with ethyl acetate. The ethyl acetateextracts are dried and the content of β-lapachone quantitated by HPLC asdescribed above.

Alternative means of determining the quantity of ketal conjugated topolymeric vehicles is described in Example 11.

Example 11 Determination of the quantity of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione that isconjugated to poly-L-glutamic acid Example 11(a)5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl glycinateγ-poly-L-glutamate conjugate

The conjugate is dissolved in a phosphate buffer adjusted to the finalpH of 9.6 at a concentration of about 0.1 mg/ml. The complete release of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione from theconjugate is achieved within 2-3 days as monitored by HPLC. In additionto the 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione peak, twoadditional peaks are detected in the HPLC chromatogram. These peaks arebelieved to be products of the degradation of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione and an alternatehydrolysis product of conjugate. The concentration of released2,2-dimethyl-3,4-dihydro-2′-1-benzo[h]chromene-5,6-dione is calculatedby comparing the peak areas with the previously obtained calibrationcurve of 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dionestandard. The concentration of the2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione is then correctedfor the degradation by taking into account the peak areas of the twodegradation products (assuming they have identical extinctioncoefficients). Loading on the polymer is then calculated as follows:

% loading=mgof(2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione)/mg ofconjugate×100

Hydrolysis of the 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dionefrom the conjugate proceeds faster at higher pH (20 min. at pH 11.4).However at this pH the degradation and the alternate hydrolysis productsare generated at higher levels, which underestimates the actual loadingon the conjugate

Direct UV method Conjugates are dissolved in DMSO at a concentration of0.1 mg/ml. The absorption of aliquots (250 μl) of the solution areanalyzed for the absorbance at 305 nm with a Spectra MAX, 96 well plateUV spectrometer (Molecular Devices). The amount of(5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylglycinate hydrochloride) loaded on the polymer is determined from acalibration curve of(5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ylglycinate hydrochloride) dissolved in DMSO. Both free and polymer bound2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione display anabsorbance maximum at 305 nm. The loadings obtained from the direct UVmethod correlated well with those determined by hydrolysis conducted atpH=9.6.

Example 11(b)N-{2-[(4-{[(6Z)-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]-chromen-6(51-1)-ylidene]amino}benzoyl)oxy]ethyl}γ-poly-L-glutamate

The conjugate is dissolved in water at a concentration of about 0.3mg/ml and sonicated to facilitate dissolution. Ten-fold dilution into 1M HCl solution almost instantly facilitates the hydrolysis of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione from theconjugate (˜4 min treatment time). The concentration of released2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione is determined byHPLC and the quantity calculated by comparing the peak areas with apreviously obtained calibration curve of2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione standard, asdescribed above in Example 10. No additional degradation peaks wereobserved in the HPLC chromatogram and the loading on the polymer wasthen calculated as above.

Example 11(c)(2,2-dimethyl-5-oxo-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′-yl)methylγ-poly-L-glutamate sodium

The conjugate is dissolved in 0.1 M NaOH solution at a concentration ofabout 1 mg/ml. Base treatment releases the4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one(A) from the conjugate in about 20 min as determined by HPLC. Theconcentration of released4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one(A) is calculated by comparing the peak areas with the previouslyobtained calibration curve of4′-(hydroxymethyl)-2,2-dimethyl-3,4-dihydro-2H,5H-spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-5-one(A) standard. The loading is expressed in2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (B) equivalentsby factoring in the molecular weight difference:

mg of B=mg of A×(Mw of B)/(Mw of A)

% loading=mg of B/mg of conjugate×100

Example 12 Cell Based Assays

Cell viability is determined by measuring the activity of dehydrogenaseenzymes in metabolically active cells using a tetrazolium compound, MTS.The assay is performed as described in Promega Technical Bulletin No.169 (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay). Twocell lines, DLD-1 and NCM-460 are assayed (see, e.g., Table 2). Cellsare maintained at 37° C., 5% CO₂. Adherent cells are maintained DMEMmedia (4.5 g/L glucose) supplemented with 15% heat-inactivated FBS, 10mM L-glutamine, and 10 mM Hepes pH 7.5. Suspension cells are maintainedin RPMI 1640 media, supplemented with 10% heat-inactivated FBS and 10 mMHepes pH 7.5. Briefly, cells are seeded in 96-well plates and incubatedfor 16-24 hours. Candidate compounds, including the polymer-modifiedtherapeutics to be tested, are serially diluted in the indicatedsolvents are, further diluted in cell culture media, and then added tocells. Cells are incubated in the presence of candidate compound for 20hours or 72 hours, as indicated. MTS stock solution (MTS 2 gm/L, PMS46.6 mg/ml in PBS) is added to the cells (final concentration MTS 2 gm/Land PMS 7.67 mg/L) and incubated for 4 hours. SDS is added to a finalconcentration of 1.4% and absorbance at 490 nM is measured within twohours using a plate reader. The IC₅₀ is defined as the concentration ofcompound that results in a 50% reduction in the number of viable cellsas compared to control wells treated with DMSO only (0.33%) and 0.3%SDS, and is calculated using non-linear regression analysis. The data isrepresented graphically in FIG. 1: Panels A-D, and the IC₅₀ values aregiven in Table 2.

TABLE 2 POLYMER MW (Da) DLD-1 NCM-460 COMPOUND LOADING INCUBATION CELLSCELLS (SOLVENT) (Weight %) TIME (Hours) IC₅₀ (μM) IC₅₀ (μM)2,2-dimethyl-3,4-dihydro- N.A. 20 3.13 8.19 2H-benzo[h]chromene-5,6-dione (DMSO) 5-(acetyloxy)-2,2- 17,100 20 4.42 8.12dimethyl-3,4-dihydro-2H- 18.6 ± 0.6 benzo[h]chromen-6-yl glycinateγ-poly-L- glutamate (PEG/water) (2,2-dimethyl-5-oxo-3,4- 17,10020 >100 >100 dihydro-2H,5H- 16.2 ± 0.1 spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′- yl)methyl γ-poly-L- glutamate sodium (water)2,2-dimethyl-3,4-dihydro- N.A. 72 1.71 3.25 2H-benzo[h]chromene-5,6-dione (DMSO) 5-(acetyloxy)-2,2- 17,100 72 1.39 3.66dimethyl-3,4-dihydro-2H- 18.6 ± 0.6 benzo[h]chromen-6-yl glycinateγ-poly-L- glutamate (PEG/water) (2,2-dimethyl-5-oxo-3,4- 17,10072 >100 >100 dihydro-2H,5H- 16.2 ± 0.1 spiro[benzo[h]chromene-6,2′-[1,3]dioxolan]-4′- yl)methyl γ-poly-L- glutamate sodium (water)N.A.: Not Applicable PEG/water: is 20% polyethylene glycol 300 Mw and80% water.

Example 13 Analytical Determination of β-Lapachone Levels in Plasma

Chromatographic analysis of plasma samples to determine plasmaβ-Lapachone levels is conducted employing a Zorbax SB C8 HPLC column(2.1×30 mm, 5μ particle size). The chromatographic system employs a CTCPAL Leap autoinjector (Leap Technologies, Carrboro, N.C.) with anAgilent (Agilent Technologies, Palo Alto, Calif.) 1100 HPLC systeminterfaced to a Micromass Quattro Ultima (Waters Corporation, Milford,Mass., USA) triple quadrapole mass spectrometer is employed to analyzesamples. Chromatographic separations were conducted employing a gradientformed from mobile phase A (0.1% formic acid (FA) in water) and mobilephase B (0.1% FA in acetonitrile). The gradient program employed for thechromatographic separation is described in Table 3. The injection volumeemployed is 10 μL. The mass spectrometer is operated using ESI(electrospray ionization) positive in MRM (multiple reaction monitoring)mode. Mass transitions are monitored, respectively, for β-lapachone-D6(Internal Standard, IS)=249>159, β-lapachone=243>159, andβ-lapachone-ketal=317>243. Argon is used as the collision gas andNitrogen is used as the desolvation gas. The collision cell gas pressureis 1.7×10⁻³ mbar. The cone gas flow is 108 L/h and the desolvation gasflow is 537 L/h. The source temperature is set to 110° C. and thedesolvation temperature is 400° C. The capillary voltage is set to 3.9kV, the cone voltage is set to 25 V, and the collision energy is 30 V.Data is processed using QuanLynx with MassLynx version 4.0 software.

TABLE 3 Gradient HPLC parameters Time (min) % B Flow (mL/min) 0 40 0.30.3 40 0.3 2.75 95 0.3 3.25 95 0.3 3.26 40 0.6 3.50 40 0.6 3.75 40 0.3

Standard stock solution of β-lapachone is prepared by dissolving 2-3 mginto an equivalent volume of DMSO to yield a 1 mg/mL stock solution.Spiking Solutions at 20,000 ng/mL are prepared by taking 20 μL of the 1mg/mL stock and combining them with 980 μL of water. Internal standard(IS) stock solution of β-lapachone-D6 is prepared by dissolving 1 mg ofβ-lapachone-D6 into 2 mL of DMSO. An IS working solution at aconcentration of 300 ng/mL is prepared from the 1 mg/mL IS stock bydilution with acetonitrile. Standard solutions for calibration areprepared by dilution of the appropriate Spiking Solution with blank NcrNU/NU mouse plasma (Bioreclamation, Inc., East Meadow, N.Y.).

Example 14 In Vitro Release of Therapeutic Agents from PolymerConjugates in Murine Plasma

Standard stock solutions of polymer-modified β-lapachone conjugates areprepared by dissolving 16.6 mg of Sample A, 5.45 mg of Sample B, 5.9 mgof Sample D, and 6.39 mg of Sample J (Table 1), respectively into 1 mLof 40 mM phosphate buffer pH 7.4 for Samples A, B and D, and 1 mL ofwater for Sample J. Based on loading factors these solutions shouldyield ˜1 mg/mL equivalents of β-lapachone upon hydrolysis. A 20,000ng/mL (β-lapachone equivalents) Spiking Solution of each is prepared bytaking 20 μL aliquots of the respective stock solution and combining itwith 980 μL of its respective diluent. A β-lapachone standard isprepared at 2000 ng/ml (a single point calibrant) by making a 1 to 10dilution of the appropriate 20,000 ng/mL Spiking Solution in blank NcrNU/NU mouse plasma (Bioreclamation, Inc., East Meadow, N.Y.); forcalibration 50 μL of the standard (single point calibrant) is combinedwith 50 μL of water mixed and 200 μL of the IS working solution isadded. This solution is mixed and then centrifuged at 13,000 rpm for 10minutes. The supernatant is transferred to a HPLC vial for analysis. Forplasma stability measurements, 50 μL of 0, 1, 2, and 5 hour of eachplasma time point sample (2000 ng/mL polymer-modified β-lapachoneconjugate in plasma) is combined with 100 μL of IS working solution,mixed and centrifuged at 13,000 rpm for 10 minutes. Control samples ofpolymer-modified β-lapachone conjugates are prepared at 2000 ng/mL(β-lapachone equivalents) by making a 1 to 10 dilution of theappropriate 20,000 ng/mL spiking solution in the appropriate diluent(either water or phosphate buffer). Control samples (2000 ng/mL) arecombined with 50 uL diluent and then 200 μL of the IS working solutionis added. The samples are mixed and then centrifuged at 13,000 rpm for10 minutes. The supernatant is transferred to a HPLC vial for analysis.

Example 15 Tumor and Plasma Levels of Free and Total β-Lapachone in NcrNU/NU Mice

Female Ncr NU/NU mice (8 weeks of age) (Charles River Laboratories) wereimplanted subcutaneously with 5 million HT29 human colon cancer cells bysubcutaneous injection in the flank. (HT-29 cells are grown inDulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetalbovine serum (FBS) and 1% penicillin/streptomycin (PS) (Roche) in a in ahumidified 5% CO2 atmosphere at 37° C.). Seven days following HT29 cellimplantation all mice are weighed prior to dosing with polymer-modifiedβ-lapachone conjugates: Sample B, 14.5 mg/ml PEG 400:50 mM phosphatebuffer pH 9.0, 139 mg/kg (26 mg/kg β-lapachone equivalents), FIG. 3;Sample J, 74 mg/ml PEG 400:50 mM phosphate buffer pH 9.0, 370 mg/kg (60mg/kg β-lapachone equivalents), FIG. 4; Sample B, 14.5 mg/ml PEG 400:50mM phosphate buffer pH 9.0, 72.5 mg/kg (14 mg/kg β-lapachoneequivalents) and Sample D, 15.1 mg/ml PEG 400:50 mM phosphate buffer pH9.0, 75.3 mg/kg (14 mg/kg β-lapachone equivalents) FIGS. 5A and 5B; byiv bolus administration (dose volume 5 ml/kg) and β-lapachone in 40%hydroxypropyl-β-cyclodextrin (60 mg/kg) by ip administration. Blood andis collected at the following time points post dosing: 0.25, 1, 2, 4, 8,and 24 hours. For blood draws, the dead volume of 1 cc syringe Fittedwith 27 gauge needle is filled with EDTA solution (1.5% w/v in 0.9%saline); total volume of EDTA solution ˜80 μl per syringe. Cardiacpuncture is then performed yielding approximately 500 μl of blood fromeach mouse. Blood samples are immediately placed on ice and centrifugedat 4° C. Plasma samples are flash frozen on dry ice and stored at −80°C. Following blood sampling, tumor samples are harvested and flashfrozen on dry ice then stored at −80° C.

A calibration curve is prepared in the range of 1 to 2000 ng/mL (1, 3,6, 10, 30, 60, 100, 300, 600, 1000, and 2000 ng/mL) by performing serialdilutions of a 2000 ng/mL standard prepared in Ncr NU/NU mouse plasma(Bioreclamation, Inc., East Meadow, N.Y.). 50 μL of each calibrationstandard is combined with 100 uL of the IS working solution, mixed andcentrifuged at 13,000 rpm for 10 minutes. The supernatant is transferredto a HPLC vial for analysis. For tumor pharmacokinetic (PK) studies thecalibration standards are prepared in the same fashion as the plasmacalibration standard with the exception that blank tumor homogenate (seesample preparation section, below) is used as the diluent.

To determine free β-lapachone plasma levels 50 μL of each plasma sampleis combined with 100 μL of IS working solution, mixed, and thencentrifuged at 13,000 rpm for 10 minutes. The supernatant is transferredto a HPLC vial for analysis. Where required, plasma samples are dilutedeither 1 to 10 or 1 to 100 in blank plasma. To determine freeβ-lapachone in tumor samples, each sample (including blank tumors forcalibration curve preparation) is homogenized (1 mg tissue to 10 μLbuffer) in 10 mM ammonium acetate buffer pH 7.3 using a 2 mL handoperated dounce homogenizer. The tumor PK samples are then prepared foranalysis in the same manner as described for the plasma samples (i.e.,50 μL samples are added to 100 μL IS working solution).

To determine total β-lapachone in plasma and tumor, samples arehydrolyzed by combining 50 μL of the sample with 50 μL of either 1 N HCl(for Sample A) or 0.1 N NaOH (for Samples B, D, and J). After allowingthe hydrolysis samples to stand for an appropriate interval of time, 50μL of each hydrolyzed sample is combined with 100 μL of IS workingsolution, mixed, and then centrifuged at 13,000 rpm for 10 minutes. Thesupernatant is transferred to a HPLC vial for analysis.

Schedule A ArQule, Inc. Assignment Recordation Ser. No. Pat. No.Application Filing Date Date Reel Frame Atty. Docket No. 11/060,746 N/A18 Feb. 2005 Jul. 5, 2005 016468 0772 22596-545 (AQ0117) 11/062,875 N/A23 Feb. 2005 May 20, 2005 016267 0275 22596-546 (AQ0119) 11/060,748 N/A18 Feb. 2005 May 20, 2005 016267 0044 22596-547 (AQ0120) 11/068,459 N/A18 Feb. 2005 Jul. 26, 2005 016573 0881 22596-548 (AQ0121) 11/060,747 N/A18 Feb. 2005 May 19, 2005 016259 0481 22596-549 (AQ0122) 10/995,565 N/A24 Nov. 2004 May 20, 2005 016267 0080 22596-550 (AQ0123) 11/060,744 N/A18 Feb. 2005 May 20, 2005 016267 0008 22596-551 (AQ0127) 11/201,170 N/A11 Aug. 2005 Mar. 8, 2006 017221 0636 22596-552 (AQ0124) 11/201,097 N/A11 Aug. 2005 Mar. 8, 2006 017272 0324 22596-553 (AQ0125) 60/914,971 N/A30 Apr. 2007 Jul. 11, 2007 019543 0483 22596-554 (AQ0133)

1. A composition comprising a carboxyl-containing polymer associated,via a linking agent of formula (II), with one or more quinone-containingtherapeutic agents, wherein said linking agent of formula (II) isassociated with said one or more quinone-containing therapeutic agentsby a quinol-ester bond; wherein said linking agent of formula (II) is ofthe form

where each R₃ and R₄ are independently selected from the groupconsisting of hydrogen, —(C₁-C₈) alkyl, —O—(C₁-C₈) alkyl, —(C₁-C₄)alkyl-aryl, aryl, and heteroaryl; R₅ is selected from the groupconsisting of hydrogen, —(C₁-C₈) alkyl, —(C₁-C₈) fluoroalkyl, aryl, andheteroaryl; R₆ is selected from the group consisting of-tert-butoxycarbonyl and carbobenzyloxy (CBZ); and m is from 1 to 8;alternatively, when m is 1, R₄ and R₅ may be taken together with thecarbon and nitrogen atoms bearing them to form a 4 to 7 memberednitrogen-containing heterocycle.
 2. The composition of claim 1, whereinsaid one or more quinone-containing therapeutic agents is β-lapachone,and said carboxyl-containing polymer is a polyethylene glycols(PEG)-dicarboxylic acid, poly(styrene-co-maleic anhydride) copolymer(SMA), poly-L-glutamic acid (PGA) or poly-L-aspartic acid (PAA).
 3. Thecomposition of claim 2, wherein said carboxyl-containing polymer is aPGA polymer, and said carboxyl-containing polymer comprises one or moreresidues of the form

where “*” indicates the points of attachment to other residues of saidPGA polymer.
 4. A pharmaceutical composition comprising atherapeutically effective amount of a composition of claim
 1. 5. Thepharmaceutical composition of claim 8, wherein said pharmaceuticalcomposition is a formulation for oral, parenteral, or intravenousadministration.
 6. A composition comprising a carboxyl-containingpolymer associated, via a linking agent of formula (II), withβ-lapachone, wherein said linking agent of formula (II) is associatedwith said β-lapachone by a quinol-ester bond.
 7. The composition ofclaim 6, wherein said carboxyl-containing polymer is a PEG-dicarboxylicacid, SMA, PGA or PAA polymer.
 8. A pharmaceutical compositioncomprising a therapeutically effective amount of a composition of claim6.
 9. The pharmaceutical composition of claim 8, wherein saidpharmaceutical composition is a formulation for oral, parenteral, orintravenous administration.